Conflicting reports on modulation of T-type calcium channels by protein tyrosine kinases (PTKs) have been published. In mouse spermatogenic cells inhibition of PTK by tyrphostin enhanced the current through the T-type calcium channels (Arnoult et al. 1997). In contrast, in NG108-15 cells inhibition of PTKs by genistein, lavendustin and herbimycin suppressed current amplitude (Morikawa et al. 1998). We have tested the effect of the PTK inhibitor genistein and its inactive analogon genistin on recombinant Ca_v3.1 (T-type) calcium channels permanently expressed in human embryonic kidney-293 cells. Calcium currents through the expressed channels were measured in whole cell patch clamp. External solutions contained (mmol/l): 2 CaCl₂, 155 NaCl, 5 CsCl, 1 MgCl₂ and 10 Hepes; pH 7.4 with NaOH. Internal solution contained (mmol/l): 130 CsCl, 5 MgCl₂, 10 TEACl, 5 NaATP and 10 Hepes; pH 7.4 with CsOH. Stock solutions (10 mmol/l) of genistein and genistin were prepared in DMSO. Final concentration of vehicle in experimental solutions was 0.5%. All values are given as mean ± S.E.M. Significance of the difference between two datasets was tested by paired and unpaired Student′s t test, as appropriate. During cell perfusion with internal solution with 0.5% DMSO, run-down of 8.0±3.7% (n=13) calcium current amplitude was observed over the time span of 12-15 min. This minor run-down was not affected by the presence of 0.5% DMSO in external solution. Extracellular application of 50 μM of genistein inhibited current amplitude by 74.8±3.4% (n=11). The effect developed slowly over the time span of 200-220 s and it was only partly reversible. Amplitude inhibition was accompanied by an acceleration of current inactivation from 13.7±1.0 ms to 11.2±0.7 ms (n=6; p<0.001) and a deceleration of current activation from 0.96±0.06 ms to 1.45±0.08 ms (n=7; p<0.001). Both effects were fully reversible. While the acceleration of inactivation had a similar time course as the amplitude inhibition, deceleration of activation developed more rapidly and saturated after approximately 80 s. During intracellular application of 50 μM of genistein current amplitude was decreased by 10.4±1.1% (n=10). This decrease was not significantly different from run-down observed in cells perfused by vehicle alone. 50 μM of genistin (genistein, 7-O-β-D-glucopyranoside) had no significant effect on current amplitude and kinetics whether it was applied extracellularly or intracellularly. We suggest that genistein inhibits Ca_v3.1 channel by a PTK-independent mechanism. This is supported by the evidence that genistein acted from outside the cell and remained ineffective when applied intracellularly. Because of its effects on current kinetics, interaction of the drug with the channel may be more complex than simple occlusion of conductive pore. Ineffectiveness of its analogon genistin could be explained by the different size of this molecule.