The aim of this investigation was to study L-type and T-type Ca2+ current (ICaL and ICaT) in short-term cultured adult guinea pig ventricular myocytes. The isolated myocytes were suspended in serum-supplemented medium up to 5 days. Using whole-cell patch clamp techniques ICaL and ICaT were studied by applying voltage protocols from different holding potentials (–40 and –90 mV). After 5 days in culture the myocytes still showed their typical rod shaped morphology but a decline in cell membrane capacitance (26 %). The peak density of ICaT was reduced significantly between day 0 (–1.60.37 pA/pF, n=9) and day 5 (–0.40.13 pA/pF, n=11), whereas peak ICaL density revealed no significant differences during culturing. The ICaT/ICaL ratio dropped from 0.13 at day 0 to 0.05 at day 5. Compared with day 0 ICaL the steady state inactivation curve of day 1, day 3 and day 5 myocytes was slightly shifted to more negative potentials. Our data indicate that guinea pig ventricular L-type and T-type Ca2+ channels are differently regulated in culture.