The effects of thyroid hormone (3,3',5-triiodo- L -thyronine, T3) on pacemaker activity were studied with electrophysiological and pharmacological approaches using spontaneously beating neonatal atrial myocytes cultured from 2-day-old rats. Treatment with T3 (10(-8)m) for 24-48 h led to a positive chronotropic effect. The beating rate of T3-treated cells was 244+/-19 beats/min and for control cells it was 122+/-10 beats/min (P<0.05). Action potentials were recorded and showed that the predominant effect of T3 was to increase the diastolic depolarization rate (99.5+/-9.8 in T3-treated group v 44.0+/-7.8 mV/s in untreated group). Some cells that exhibited pacemaker activity lacked a pacemaker current (I(f)) under voltage clamp conditions I(f)was recorded in 5 of 12 spontaneously active control cells and in 6 of 10 T3-treated cells. In those cells exhibiting the pacemaker current, the I(f)density was significantly larger in T3-treated cells (-7.9+/-2.6 pA/pF v-1.8+/-0.5 pA/pF in control). The L-type Ca2+ current density was similar in the two groups (at -7 mV, -7.5+/-1.5 in treated group v-8.6+/-1.0 pA/pF in control). In the presence of T3, the Na+-Ca2+ exchanger current (I(Na/Ca)) density was larger (e.g. at +60 mV, it was 4.8+/-0.5 v 3.5+/-0.2 pA/pF in control cells, P<0.05). As intracellular Ca2+ is extruded from the cell, the electrogenic Na+-Ca2+ exchanger causes a declining inward current, which may contribute to the pacemaker potential-this declining inward current was demonstrated using the action potential voltage clamp technique and was shown to be larger in T3-treated myocytes. Our data demonstrate that thyroid hormone enhances pacemaker activity and that this may be due in part to an increased Na+-Ca2+ exchanger activity.