We determined the relative effects of acetylcholine (ACh) and oxotremorine (Oxo) on sinoatrial and atrioventricular nodal function in the fetal heart. Oxo is a cholinesterase-resistant agonist. Hearts from fetal guinea pigs in the last trimester were perfused retrogradely through their coronary vasculature with a physiologic salt solution containing ACh or Oxo, 1×10−8 to 2.5×10−7M. We measured sinoatrial (SA) rate; atrioventricular conduction time (AVCT); and the Wenckebach cycle length (WBCL), the shortest paced cycle length resulting in 1:1 AV conduction. Control values were (X±SE, n=8):SA rate, 196±15 beats per min; AVCT, 81±3 msec; WBCL, 208±11 msec. Both ACh and Oxo induced concentration dependent, reversible changes in all 3 variables. Maximum effects of ACh occurred at 2.5×10−7M; those of Oxo between 1×10−8 and 5×10−8M. Maximum percent change, compared to control, of ACh and Oxo were, respectively, -28±3% and -51±7% for SA rate (p<.01); +10±6% and +23±2% for AVCT (p<.02); and +34±9% and +1141±48% (p<.02) for WBCL. These data indicate that Oxo is a more potent agonist than ACh with respect to its electrophysiologic effects on the fetal heart. These differences may result from a relatively high cholinesterase activity which attenuates the action of ACh but not of Oxo.