Recent studies have documented that testosterone relaxes several smooth muscles by modulating K+ channel activities. Smooth muscles of seminal vesicles play a fundamental role in ejaculation, which might involve testosterone. This study was aimed to assess the role of testosterone in seminal vesicular motility by studying its effects on contractile agents and on the ion channels of single vesicular myocytes in a rabbit model. The contractile responses of circular smooth muscle strips of rabbit seminal vesicles to norepinephrine (10ՌM), a high concentration of KCl (70 mM), and testosterone (10ՌM) were observed. Single vesicular myocytes of rabbit were isolated using proteolytic enzymes including collagenase and papain. Inside-out, attached, and whole-cell configurations were examined using the patch clamp technique. The applications of 10ՌM norepinephrine or 70 mM KCl induced tonic contractions, and 10ՌM testosterone (pharmacological concentration) evoked dose-dependent relaxations of these precontracted strips. Various K+ channel blockers, such as tetraethylammonium (TEA; 10 mM), iberiotoxin (0.1ՌM), 4-aminopyridine (4-AP, 10ՌM), or glibenclamide (10ՌM) rarely affected these relaxations. Single channel data (of inside-out and attached configurations) of BK channel activity were also hardly affected by testosterone (10ՌM). On the other hand, however, testosterone reduced L-type Ca2+ currents significantly, and found to induce acute relaxation of seminal vesicular smooth muscle and this was mediated, at least in part, by Ca2+ current inhibition in rabbit.