1. External application of the unsaturated fatty acid arachidonic acid (AA) to frog ventricular cells caused a large inhibition (approximately 85%) of the L-type calcium current (ICa,L) previously stimulated by the beta-adrenergic agonist isoprenaline (Iso). The concentration producing half-maximal inhibition (K1/2) was 1.52 microM. The inhibitory effect did not affect the peak current-voltage relationship but produced a negative shift in the inactivation curve. 2. The inhibitory effect of AA also occurred in cells internally perfused with cAMP and non-hydrolysable analogues of cAMP. These data suggest that AA is acting by a mechanism located beyond adenylyl cyclase and does not involve changes in intracellular cAMP levels. 3. AA also inhibited the calcium current stimulated by internal perfusion with the catalytic subunit of protein kinase A (PKA), suggesting that AA acts downstream of channel phosphorylation. 4. The inhibitory effect of AA on the isoprenaline- or cAMP-stimulated ICa,L is largely reduced in cells internally perfused with the thiophosphate donor analogue of ATP, ATP gamma S, or protein phosphatase 1 and 2A inhibitors like microcystin (MC) or okadaic acid (OA). External application of the phosphatase inhibitor calyculin (Caly) also reduced the AA effect. These data suggested that the AA effect on ICa,L involves activation of protein phosphatase activity. 5. The effect of AA on ICa,L was not affected by staurosporine, an inhibitor of protein kinases. It was also unaffected in cells internally perfused with GTP gamma S. These results suggest that neither a PKC- nor a G-protein-mediated mechanism are likely to be involved in the effect of AA on ICa,L. 6. A saturated fatty acid, myristic acid (MA), had no inhibitory effect on the isoprenaline-stimulated Ca2+ current, whereas, in the same cells arachidonic acid produced approximately 85% inhibition of ICa,L. 7. The inhibitory effect of AA was not affected by exposing the cells to indomethacin (Indo), an inhibitor of the metabolism of AA by cyclo-oxygenase, nor nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. However, the non-metabolizable analogue of AA, 5,8,11,14-eicosatetraynoic acid (ETYA), was without effect on the isoprenaline-stimulated ICa,L. 8. These results suggest that AA inhibits ICa,L via a mechanism which involves, in part, stimulation of protein phosphatase activity. This process could provide a new mechanism in the modulation of calcium channel activity.