We have previously shown an increase in arachidonic acid (AA) release in response to proinflammatory cytokines in adult rat ventricular myocytes (ARVM). AA is known to alter channel activities; however, its effects on cardiac L-type Ca²⁺ channel current (I_Ca,L) and excitation-contraction coupling remain unclear. The present study examined effects of AA on I_Ca,L using the whole-cell patch-clamp technique and on cell shortening (CS) and the Ca²⁺ transient of ARVM. I_Ca,L was monitored in myocytes held at -70 mV and internally dialyzed and externally perfused with Na⁺- and K⁺-free solutions. Exposure to AA caused a voltage-dependent block of I_Ca,L concentration-dependently (IC₅₀ of 8.5 µM). The AA-induced inhibition of I_Ca,L is consistent with its hyperpolarizing shift in the voltage-dependent properties and reduction in maximum slope conductance. In the presence of AA, BSA completely blocked the AA-induced suppression of I_Ca,L and CS. Intracellular dialysis with AA had no effect on the current density but caused a small depolarizing shift in I_Ca,L activation curve, suggesting a site-specific action of AA. Moreover, intracellular AA had no effect on the extracellular AA-induced decrease in I_Ca,L. Pretreatment with indomethacin, an inhibitor of cyclo-oxygenase, or addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase, had also no effect on AA-induced changes in I_Ca,L. Furthermore, AA suppressed CS and Ca²⁺ transients of intact ARVM with no significant effect on SR function and myofilament Ca²⁺ sensitivity. Therefore, these results suggest that AA inhibits contractile function of ARVM, primarily due to its direct inhibition of I_Ca,L at the extracellular site.