The in vitro dose-relaxation curves of four isomeric organic mononitrates: L-isoidide mononitrate (L-IIMN), isosorbide-2-mononitrate (IS-2-MN), isomannide mononitrate (IMMN), and isosorbide-5-mononitrate (IS-5-MN), were determined with rat aorta rings. These mononitrates relaxed vascular tissue in a concentration-dependent manner. Based upon the EC50 obtained from the Hill equation, the relative potency of L-IIMN: IS-2-MN: IMMN: IS-5-MN was 43.2:12.2:2.3:1. The Hill exponential coefficients were identical (value of about 1.5) in these four isomeric mononitrates, suggesting that they are likely to have a common mechanism of action. For all four isomers, relaxation was fairly immediate after addition of the tested compound into the tissue bath, with a 2- to 3-min-delay to reach steady-state effect. The rates of relaxation were then used to construct a pharmacodynamic model that described the time course of relaxation for these compounds. This theoretical analysis suggested that in vitro nitrate action is mediated by a catenary process, consistent with published biochemical evidence that suggests a series of reactions involving metabolic activation to nitric oxide, production of cyclic GMP, and myosin light-chain phosphorylation to produce relaxation. Via this pharmacodynamic model, the half-lives of nitric oxide and cGMP in the smooth muscle cells were estimated to be 15.2 and 23.1 sec, respectively, consistent with literature reports. Results from the present study indicated the potential use of in vitro pharmacodynamic modeling in confirming mechanism(s) of drug action obtained through biochemical or other methods.