A low-density primary culture of trout ventricular myocytes in serum-free growth medium was established and maintained for up to 10 days at 17°C. The myocytes retained their normal rod shaped morphology, capacitive surface area of the sarcolemma (SL), and contractile quiescence. However, sarcolemmal cation currents changed significantly, some permanently, some transiently, after 8–10 days of culture. TTX-sensitive sodium current ( INa) and Ba2+-sensitive background inward rectifier potassium current ( IK1) were permanently depressed to 24–28% of their control density measured in freshly isolated myocytes. In contrast, L-type calcium current ( ICa) was only transiently downregulated; after 2–3 days in culture, the density of the current was 32% of the control and recovered to the control value after 8–10 days in culture. The changes in membrane currents were reflected in the shape of the action potential (AP). After 2–3 days in culture, maximal overshoot potential and resting potential were significantly reduced, and the durations of the AP at 50 and 90% repolarization were significantly increased. These changes became significantly more pronounced after 8–10 days of culture, with the exception of AP duration at 50% repolarization level. The shortening of the early plateau phase may reflect an additional change to an outward current, presumably the rapid component of the delayed rectifier ( IKr). Although the present findings indicate that fish cardiac myocytes can be maintained in serum-free primary culture for at least 10 days at 17°C, some but not all of the electrophysiological characteristics of the myocytes change markedly during culture. The changes in ion currents were not due to loss of sarcolemmal membrane and therefore are likely to represent altered expression of cation currents as an adaptive response to culture conditions.