Testosterone (T) acts as a coronary vasodilator and reduces myocardial ischemia in men with coronary heart disease. We have previously demonstrated that T inhibits the pore forming alpha1c subunit of the cardiovascular L-type calcium channel in HEK293 cells. In this study we investigated the effects of T and 5β-dihydrotestosterone (5β-DHT) on extracellular calcium entry in A7r5 vascular smooth muscle cells (VSMCs). A7r5 cells grown on coverslips in 12-well plates in DMEM +10% foetal bovine serum were incubated in medium containing the calcium fluorescence probe Fura2-AM 4 μM for 40-min, at 37 degC. Coverslip fragments were placed in a perfusion chamber and changes in calcium were indicated by fluorescence emitted at 510 nm after excitation at 340 and 380 nm, in the presence of test substances. High potassium (K+) induced a change in cellular fluorescence of 0.12±0.02 ratio units. Subsequent recordings are expressed as a percentage of this response. Compared to ethanol (0.1%), 2-min incubation with T (1, 3, 10, 100 nM) caused a concentration-dependent inhibition of this response; 94.2±7.4%K+, 98.4±5.1%K+, 67.6±4.0%K+ (P<0.01), 51.5±5.8%K+40.9±2.6%K+ all P<0.001 respectively. IC50=3.1 nM. 2-min incubation with 5β-DHT (1, 10, 100 nM) also caused an inhibition of this response; 58.3±9.8%K+ (P<0.01), 44.9±2.5%K+, 39.3±6.1%K+ (both P<0.001). Incubation with nifedipine (L-type calcium-channel blocker) (5 μM) also caused similar inhibition of this response; 46.7±4.1%K+ (P<0.001), as did pimozide (T-Type calcium channel blocker) (1μM) 31.3±4.4%K+ (P<0.001) and calcium-free buffer almost abolished the response 6.5±1.3%K+ (P<0.001), as did co-incubation with nifedipine (5 μM) + pimozide (1 μM) 12.3±1.3%K+ (P<0.001). Co-incubation with T (10 nM) + nifedipine (5 μM) showed no extra inhibition; 44.1±6.8%K+ (P<0.001) compared to nifedipine and T only. We conclude that physiological concentrations of T and 5β-DHT inhibit extracellular calcium entry via L-type voltage-gated calcium channels in VSMCs via a non-genomic manner, with an effect similar to that of nifedipine.