Structure-activity studies with a number of adenosine derivatives and analogs, measuring their relaxant effects in a variety of smooth muscle systems, were conducted in the hope of obtaining indications of the possible involvement of adenylate cyclase in their mechanism of action. While it was confirmed that a C6 aminofunction is of importance for agonist activity, several compounds, in particular the relatively potent N6-hydroxylaminopurine ribonucleoside, were not antagonized by 8-p-sulfophenyltheophylline, indicating that some nucleosides cause smooth muscle relaxation by a mechanism other than adenosine receptor stimulation. Nucleosides not bearing a C6 aminofunction were essentially inactive in rabbit intestine but showed weak relaxant effects in bovine coronary artery; this may indicate a difference between the adenosine receptor systems in these tissues and the intracellular mechanisms of relaxation. Comparing the relative potencies of compounds such as adenosine, 2-chloroadenosine, 5'-(N-ethylcarboxamido)adenosine, and (-)N6-(R-phenylisopropyl)adenosine, which have been used widely to classify adenylate cyclase-coupled adenosine receptors, no uniform pattern became apparent among different smooth muscle systems used in this study and reported in the recent literature. Thus, we conclude that a classification of smooth muscle adenosine receptors according to criteria established for cyclase-coupled receptors may be inappropriate or misleading, particularly with respect to implications of adenylate cyclase involvement in the relaxant effects of adenosine and related nucleosides.