Hormones can regulate cardiac L-type Ca2+ channels via cAMP-dependent protein kinase (PKA) phosphorylation. However, regulation of the cloned L-type Ca2+ channel has been difficult to demonstrate conclusively. We stably transfected a human embryonic kidney (HEK-293) cell with the cardiac alpha 1 and beta 2 subunits, then examined PKA modulation of the Ca2+ current. Although forskolin did not increase basal Ca2+ current, the PKA inhibitors, H-89 and Rp-cAMPS, could inhibit basal current. We reversed H-89 inhibition with either forskolin or okadaic acid. We conclude that the channel was phosphorylated under basal conditions, and that inhibition of PKA allowed dephosphorylation. These studies demonstrate that reversible PKA regulation of cloned Ca2+ channels can be studied in HEK-293 cells.