This study examinedin vitromyocardial depression by 50% N2O. Maximal isometric contractions of guinea pig right ventricular papillary muscles were studied in Tyrode's superfusate at 37° C within a gas-tight chamber. Superfusate (pH at 7.45) and chamber were equilibrated with 95% O2/5% CO2. After control measurements in 95% O2, muscles were studied with 50% N2and 50% N2O (45% O2/5% CO2) in random order with an intervening and final recovery in oxygen. Muscles were field stimulated after rest and at 0.1–3 Hz. At 37° C, muscle performance deteriorated over time with exposure to reduced oxygen; therefore, identical experiments were performed at 30° C in which no systematic deterioration occurred. Peak tension and maximum rate of tension development (dT/dtmax) were compared for each stimulation rate. At both temperatures, N2O caused a 10–15% depression of contractility as compared to that observed with nitrogen. In a second protocol, muscles were studied at 37° C in 26 mM K+Tyrode's solution with 0.10 μM isoproterenol to study enhanced contractions mediated by slow (Ca2+-channel-dependent) action potentials. Rested-state double stimulations were used (stimulus interval, 250–600 ms) resulting in a first rested-state contraction followed by a second contraction (C2) with rapid initial tension development. The muscles were exposed to nitrogen and N2O as in the force-frequency experiments and did not deteriorate over time. In this setting, N2O also caused a 10–15% depression of C2 contractility as compared with nitrogen. Another set of muscles was studied in 95% O2to which 0.5% halothane or 1% isoflurane was added before exposure to nitrogen and N2O. The combined depressant action of N2O with either halothane or isoflurane did not differ from that predicted by the simple addition of independent effects; there was no evidence of synergism. Furthermore, N2O (50%) alone depressed dT/dtmaxin a manner similar to that of 0.5% halothane and different from that of 1.0% isoflurane. Experiments conducted in iso-osmolar 40 mM Na+Tyrode's solution, in which activator Ca2+arose from the sarcoplasmic reticulum Ca2+, also showed greater depression by N2O than nitrogen. N2O (50%) is a myocardial depressant independent of concurrent hypoxic effects with a pattern and magnitude of contractile depression similar to that of 0.5% halothane.