1. Free intracellular calcium concentration ([Ca2+]i) was recorded in single smooth muscle cells of the guinea-pig urinary bladder held under voltage clamp at 36 degrees C and 3.6 mM-extracellular Ca2+. The Ca2+ indicator Indo-1 was loaded into the cells through patch electrodes. To separate Ca2+ currents (ICa), superimposed K+ currents were suppressed with a Cs(+)-containing electrode solution. 2. At a holding potential of -60 mV, resting [Ca2+]i was 114 +/- 22 nM (mean +/- S.D.). During 160 ms depolarization steps to 0 mV, [Ca2+]i rose to 885 +/- 140 nM. With steps of varied duration, peak [Ca2+]i increased with the time of depolarization up to about 1 s. Upon repolarization [Ca2+]i recovered to resting levels with a half-decay time of about 1 s; recovery was not significantly changed with repolarization potentials between -50 and -100 mV. 3. The potential dependence of the above peak [Ca2+]i transients was bell shaped, with a threshold around -40 mV and a maximum at 0 mV. During depolarization steps to potentials more positive than +80 mV [Ca2+]i did not significantly rise. 4. During step depolarizations to 0 mV lasting 10 s or longer, [Ca2+]i peaked within 814 +/- 18 ms and then decayed to a sustained level of 250 +/- 60 nM. The amplitude of the [Ca2+]i peak as well as the time course of the transient depended on the amplitude of ICa. The depolarizations increased [Ca2+]i to a sustained level with no clearly defined peak when ICa was reduced by partial inactivation or during steps close to the threshold of ICa (-40 mV). 5. The sustained level of [Ca2+]i with longer depolarizations of several seconds showed a bell-shaped voltage dependence with a maximum close to 0 mV. A bell-shaped voltage dependence for [Ca2+]i was also found during ramp-like depolarizations. However, when the rate of depolarization was low (7.5 mV s-1), the peak [Ca2+]i was found at more negative potentials (-15 mV). 6. The results are compatible with the idea that Ca2+ influx through voltage-operated Ca2+ channels is the key event in depolarization-mediated changes in [Ca2+]i in smooth muscle cells from urinary bladder.