The presence of a myocardial depressant in the plasma of canines subjected to hemorrhagic shock or derived from homogenates of canine pancreas subjected to shock-like conditions was confirmed with in vitro preparations of both isometric and isotonic contracting young canine papillary muscles. A system was developed to bioassay whole fresh plasma. Shock plasma, but not control plasma, significantly depressed the rate of muscle shortening (Vc) and relaxation (Vr) but did not alter maximum isotonic load (Po). Homogenates of canine pancreas which had been incubated under hypoxic conditions were fractionated by gel chromatography. Depressant activity was localized in a peak with a molecular weight between 500-1000. The peak from shock but not control homogenate depressed peak developed tension (AT), the rate of tension developed (+dT/dt), relaxation (-dT/dt), latency (LAT), and the time to maximum velocity of relaxation (TMVR). The effects of temperature and frequency stimulation and the influence of these factors on the contractile function of isolated myocardium in response to the depressant peak were evaluated. Muscle function was improved by cooling or increasing the frequency of stimulation. Cooling increases AT and shortens LAT, time to maximum rate of developed tension (+dT/dt), the time to peak tension (TPT), the time to maximum rate of decline in tension (-dT/dt), and duration of contraction (DUR). Cooling from 37C to 27C antagonized the depression of Po due to the shock material at either frequency (F) tested (12 or 60/minute). A faster frequency of stimulation antagonized the depressant effect on Po at 37C and both Vc and Vr at 27C. Higher frequency also antagonized the slowing effect on all time intervals due to the depressant. Relaxation was the parameter most consistently and significantly affected by the depressant factor and may be an important criterion for evaluating the role and mechanism of action of endogenous myocardial depressant factors. It can be concluded that altered temperature or frequency of an isolated tissue preparation can affect the response to an endogenous myocardial depressant and may explain failure of other laboratories to detect such a depressant.