We have examined the contribution of L-type Ca²⁺current ( I_Ca) to the activation of contraction in trout atrial myocytes under basal and phosphorylating conditions. The average myocyte length was 197 ± 14 μm, width was 5.5 ± 0.2 μm, and cell capacitance was 36.2 ± 2.2 pF. With 25 μM EGTA in the patch pipette and a stimulation frequency of 0.125 Hz, I_Cawas 2.6 ± 0.4 pA/pF and it carried a total charge of 0.10 ± 0.01 pC/pF, giving rise to a contraction of 15.2 ± 2.8% of the resting cell length. With a cell volume of 2.4 ± 0.3 pl, the charge carried by I_Cacorresponded to 14.7 ± 2.2 μmol Ca²⁺/l nonmitochondrial cell volume (μM). This can account for only 30–40% of the Ca²⁺binding to the myofilaments during a contraction. Increasing the stimulation frequency from 0.25 to 2 Hz decreased I_Caamplitude and charge by 66 ± 5 and 80 ± 3%, respectively. Elevating the pipette EGTA concentration from 25 μM to 5 mM increased I_Caamplitude and charge by ∼290%. Both isoproterenol and cAMP increased I_Caby ∼230%. The total charge carried by the isoproterenol- or cAMP-stimulated current was increased by 170%. We conclude that the use of high-EGTA concentration may overestimate the total Ca²⁺carried by I_Caunder physiological conditions. Furthermore, the results suggest that, in contrast to previous reports from other lower vertebrates, Ca²⁺flux through L-type Ca²⁺channels alone is not sufficient to fully activate contraction in trout atrial myocytes at room temperature.