Little is known about the signaling pathways involved in alpha 2-adrenergic receptor-mediated contraction of vascular smooth muscle. In the present study, we measured intracellular Ca2+ ([Ca2+]i), myosin light chain (MLC) phosphorylation, and myofilament Ca2+ sensitivity during stimulation with the relatively selective alpha 2-agonist UK 14304. These effects were compared and contrasted with corresponding changes during depolarization by elevation of the [K+] in the bathing medium. These studies were performed using spiral strips of the rabbit saphenous vein, a tissue with a relatively high density of postsynaptic alpha 2-receptors. UK 14304 (10(-5) M) caused parallel changes in [Ca2+]i, MLC phosphorylation, and force consisting of an initial phasic, followed by a sustained steady-state response. The steady-state increase in [Ca2+]i, MLC phosphorylation, and force caused by UK 14304 in the presence of 2.5 mM extracellular Ca2+ were indistinguishable from those during 51 mM K+ depolarization. However, when extracellular Ca2+ was removed in the presence of UK 14304, [Ca2+]i and MLC phosphorylation fell to resting levels, but force remained significantly elevated above basal levels. UK 14304 caused no change in the steady-state [Ca2+]i-MLC phosphorylation relation. Thus, the [Ca2+]i sensitization of force was not caused by a sensitization of MLC phosphorylation. These results indicate that in a 2.5-mM Ca2+ bathing medium, the dominant mechanism by which alpha 2-adrenergic receptor stimulation causes an increase in vascular tone is through a relatively large increase in [Ca2+]i and MLC phosphorylation. However, in Ca(2+)-free bathing medium, a second mechanism is unmasked which appears to involve an increased Ca2+ sensitivity and is independent of myosin phosphorylation.