Fatty acids acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. Recent studies show that fatty acids elevate intracellular free Ca²⁺ and increase voltage-gated Ca²⁺ current in mouse cells, although the mechanisms involved are poorly understood. We utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca²⁺ channels (LTCC) and demonstrate that cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type ₁ subunits (-cell Ca_v1.3 or lung Ca_v1.2) and ERFP-tagged islet subunits (i_2a or i₃). In COS-7 cells, elevations in intracellular Ca²⁺ mediated by LTCC were enhanced by an oleate/BSA complex. To extend these findings, Ca²⁺ current was measured in LTCC-expressing HEK 293 cells which revealed an increase in peak Ca²⁺ current within 2 minutes after addition of the oleate complex, with maximal potentiation occurring at voltages < 0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate and the presence of different subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by pretreating cells with triacsin C, suggesting that long chain CoA synthesis is necessary for Ca²⁺ channel modulation. These results show for the first time that two L-type Ca²⁺ channels expressed in -cells (Ca_v1.3 and Ca_v1.2) appear to be targeted by non-esterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.