Effects of cardiac-tissue extract on the activity of L-type Ca2+ channels were investigated in guinea-pig ventricular myocytes with the patch-clamp method. In most patches, Ca2+-channel current recorded with a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644 ran down within 5 min after excision of the patches into a solution containing EGTA. This run-down of Ca2+ channels was prevented when patches were excised into a solution containing a supernatant fraction of homogenate of guinea-pig or bovine heart. Furthermore, this tissue extract was able to restore channel activity after run-down. This channel-activating effect of the extract was abolished by heat treatment or trypsin digestion. Fractionation of the extract by gel filtration suggested that the channel-activating factor(s) had an apparent molecular weight of 2-3 x 10(5). These results suggest that some cytoplasmic protein(s) maintains the activity of the cardiac L-type Ca2+ channel.