1. We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr-HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr-HEK were characterized and compared with the properties of a heterologously expressed chimeric L-type calcium channel construct. 2. ISr-HEK activated on depolarization to voltages positive of -40 mV. It had transient current kinetics with a time to peak of 16 +/- 1.4 ms (n = 7) and an inactivation times constant of 52 +/- 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was -19.0 +/- 1.5 mV (n = 7) and the voltage for half maximal steady-state inactivation was -39.7 +/- 2.3 mV (n = 7). 3. Block of ISr-HEK by the dihydropyridine isradipine was not stereoselective; 1 microM (+) and (-)-isradipine inhibited the current by 30 +/- 4% (n = 3) and 29 +/- 2% (n = 4) respectively. (+)-Isradipine and (-)-isradipine (10 microM) inhibited ISr-HEK by 89 +/- 4% (n = 5) and 88 +/- 8% (n = 3) respectively. The 7-bromo substituted (+/-)-isradipine (VO2605, 10 microM) which is almost inactive on L-type calcium channels also inhibited ISr-HEK (83 +/- 9%, n = 3) as was observed for 10 microM (-)-nimodipine (78 +/- 6%, n = 5). Interestingly, 10 microM (+/-)-Bay K 8644 (n = 5) had no effect on the current. ISr-HEK was only slightly inhibited by the cone snail toxins omega-CTx GVIA (1 microM, inhibition by 17 +/- 3%, n = 4) and omega-CTx MVIIC (1 microM, inhibition by 20 +/- 3%, n = 4). The funnel web spider toxin omega-Aga IVA (200 nM) inhibited ISr-HEK by 19 +/- 2%, n = 4). 4. In cells expressing ISr-HEK, maximum inward current densities of 0.24 +/- 0.03 pA/pF and 0.39 +/- 0.7 pA/ pF (at a test potential of -10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 +/- 0.18 pA/pF or 1.11 +/- 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum-free medium. 5. Co-expression of a chimeric L-type calcium channel construct revealed that ISr-HEK and L-type calcium channel currents could be distinguished by their different voltage-dependencies and current kinetics. The current density after heterologous expression of the L-type alpha 1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 +/- 0.45 pA/pF) than that of ISr-HEK under the same conditions.