The discovery of EDRF caused an explosion in research, with many different groups around the world making important contributions to the search for its identity. But the realization that EDRF and NO were one and the same substance took six years of intense research. From 1980 to 1986 reports of similarities between the two gradually mounted. In hindsight this may seem to have been an inevitable accumulation of data, but at the time the picture was quite confusing. NO is an extremely reactive free radical--a result of the molecule's unpaired electron in its outer electron "shell." This high reactivity meant that scientists trying to zero in on EDRF often unwittingly perturbed NO levels when they were trying to shut off other molecular pathways, leading the researchers to the erroneous conclusion that these other pathways were responsible for producing EDRF. Moreover, NO itself seemed a poor candidate for cellular messenger. Its reaction with oxygen, for example, leads to the formation of the corrosive gas nitrogen dioxide (NO2), which is readily converted to nitric acid. (Note that NO and NO2 are distinct from nitrous oxide or N2O, the "laughing gas" used as an anesthetic by dentists.) No previously known biological signaling molecule was a free radical, let alone a radical that was a poisonous gas.

But then there were the mounting coincidences. For starters, EDRF and NO both caused blood vessel dilation, and both did so by turning on GC. This and other evidence led Ferid Murad to propose in 1986 that EDRF could be considered an "endogenous nitrate." The decisive experiments that identified EDRF as NO were performed independently by Ignarro at Tulane and the University of California, Los Angeles, by Furchgott at SUNY, and by Salvador Moncada at the Wellcome Research Laboratories in Beckenham, England. All three researchers found that NO and EDRF both decayed in a matter of seconds, were stabilized by the same conditions, and were turned off by the same battery of chemical treatments. In addition, Ignarro found that NO and EDRF underwent identical reactions with a complex chemical--an unlikely occurrence unless NO and EDRF were identical. Thus, EDRF was chemically identified to be NO.

Ignarro and Furchgott presented their results to a skeptical audience at a conference at the Mayo Clinic, in Rochester, Minnesota, in July 1986. Ignarro felt that not "a single person" in the audience believed them, but when the data were published in both 1987 and 1988, opinion swung their way. Moncada clinched the argument in an important 1987 paper. In this widely cited article he unambiguously showed that NO was made by endothelial cells. First, he measured the amount of NO produced by a known relaxant (bradykinin) acting on cultured endothelial cells. Then he added exactly that amount of NO to a blood vessel and showed that the added NO could cause a full relaxant response. Thus, the actions of NO could explain the actions of EDRF. A commentary accompanying Moncada's paper described these findings as "the climax of one of the most exciting sagas in vascular physiology and pharmacology."