We have reported previously that derivatives of adenosine cyclic 3':5'-monophosphate dramatically stimulate the activity of plasminogen activator (PA), an arginine-specific serine protease, in HTC rat hepatoma cells. We report here that these derivatives also cause striking alterations in hepatoma tissue culture cell morphology. Because PA has been shown to alter cell morphology in other cell lines, we investigated whether the morphological changes induced by cyclic nucleotides were mediated by the stimulation of PA activity. Alterations in PA activity, measured by the plasminogen-dependent solubilization of 125I-labeled fibrin, and in cell morphology, detected by evaluation of cell flattening and process extension with phase-contrast microscopy, were assessed in the same cultures under various experimental conditions. Several lines of evidence clearly dissociate these two adenosine cyclic 3':5'-monophosphate-mediated phenomena. (a) The morphological changes precede increases in either cell-associated or extracellular PA activity. (b) Upon removal of the effectors, the morphological effects are completely reversed at a time when PA activity is still considerably elevated. (c) when protein synthesis is inhibited by the addition of cycloheximide, the stimulation of PA activity by cyclic nucleotides is blocked completely, whereas the induction of morphological alterations still occurs. (d) An exogenous PA, urokinase, does not elicit the characteristic changes in cell shape. We conclude that the morphological alterations induced by adenosine cyclic 3':5'-monophosphate derivatives in HTC cells are not mediated by the stimulation of PA activity and that these two membrane-associated properties are regulated independently.