The hypothesis that 1,25-dihydroxyvitamin D3[l,25-(OH)2vitamin D3] modulates vascular smooth muscle contractile function was tested. 1,25-(OH)2vitamin D3(50 ng/day) was administered by intraperitoneal injection over a 3-day period to 13–15-week-old male spontaneously hypertensive and Wistar-Kyoto normotensive rats. On the fourth day, serum was prepared and contractile force generation of isolated mesenteric resistance arteries was examined. Treatment with 1,25-(OH)2vitamin D3approximately doubled serum levels of the hormone and increased ionized and total serum Ca2+and phosphate by 5–10%. No effect on blood pressure was detected. 1,25-(OH)2vitamin D3injection in both strains enhanced maximal stress generation to norepinephrine and serotonin by 30–40%, with no effect on apparent sensitivity of the vessels to the agonists. To assess the effect of a maneuver that elevates serum ionized Ca2+without the addition of exogenous hormone, maximal stress generation was examined in resistance arteries isolated from rats fed diets containing 0.5% or 2% calcium over a 6–7-week period. Maximal stress generation in response to norepinephrine was greater in vessels from rats of both strains maintained on 0.5% calcium. It is concluded that 72-hour in vivo treatment with 1,25-(OH)2vitamin D3increases contractile force-generating capacity of resistance arteries without affecting blood pressure. It is proposed that this action of 1,25-(OH)2vitamin D3is the result of a direct action of the hormone on the vascular wall.