The role of voltage-dependent calcium channels (VDCCs) for diameter regulation in rat mesenteric terminal arterioles (RMTA) has been studied using intra-vital microscopy. However, there is a need to develop preparations for studying vasomotor signals in isolated arterioles and in single cells isolated therefrom. Arterioles were isolated from the mesentery of humanely killed rats using enzyme digestion and mechanical dispersion. These non-pressurized microvessels had one continuous layer of smooth muscle cells in a circular arrangement around the vessel lumen. The external diameter was 31±1.2 μm (mean±S.E.M.; n = 27) and the internal (lumenal) diameter was 17±1.3 μm (n = 7), thus classifying them as terminal arterioles (lumen <50 μm). Isolated arterioles were mounted on cover slips, loaded with the Ca²⁺ indicator Fura-PE3, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was estimated as F₃₄₀/F₃₈₀. Voltage-dependent Ca²⁺ influx was evaluated as % changes from baseline of the F₃₄₀/F₃₈₀ during sustained depolarization using 75 mM bath KCl (hi-K) (Jensen et al, 2004). The overall baseline F₃₄₀/F₃₈₀ of 0.91±0.02 increased to 1.08±0.04 with hi-K solution (118.6±2.5 % increase relative to baseline, P<0.001, n = 27). Initially, we tested the effect of various modulators of L-type VDCCs. The dihydropyridine nitrendipine (10 μM), a specific L-type blocker, non-significantly changed the Ca²⁺ increase to hi-K from 128.5±6.2 % to 125.3±6.5 % relative to the baseline F₃₄₀/F₃₈₀ (N.S., n = 7). Bay-K8644 (1 μM), an L-type channel opener, did not change the resting [Ca²⁺]_i, but induced a non-significant change of the Ca²⁺ increase to hi-K from 114.8±3.1 % to 120.6±3.3 % relative to baseline (N.S., n = 5). The phenylalkylamine D-600 (2 μM) did not change the baseline ratio, yet it reduced the increase to hi-K from 113.3±2.8 % to 103.6±0.5 % relative to baseline (70±5 % inhibition, P<0.05, n = 4). NiCl₂ (500 μM), a fast and reversible inorganic blocker of T-type Ca²⁺ channels, reduced the baseline from 100 % to 97.5±0.4 % (P<0.01, n = 5), and reduced the Ca²⁺ increase to hi-K from 109.3±1.5 % to 102.1±0.1 % relative to baseline (79±2 % inhibition, P=0.001). We conclude that this arteriolar preparation is suitable for measurements of [Ca²⁺]_i or whole-cell Ca²⁺ current following single cell dispersion. Although the presence of L-type VDCCs could not be excluded, the combined dihydropyridine insensitivity and Ni²⁺ sensitivity suggests that the voltage-dependent Ca²⁺ influx in isolated RMTA is carried by channels with a T-type pharmacology, thus confirming our previous investigations (Morita et al, 2002; Jensen et al, 2004). Of note is the effect of D-600 in RMTA, suggesting that its antihypertensive capacity may partially stem from inhibition of a T-type channel in the peripheral vasculature.