Phosphorylation of the 20 kDa myosin regulatory light chains (LC₂₀) plays a key role in the regulation of smooth muscle contraction. The level of LC₂₀ phosphorylation is governed by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP) pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of LC₂₀ phosphorylation in the renal microcirculation. The available pharmacological probes are often non-specific and current techniques to directly measure LC₂₀ phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound Mn²⁺-phosphate-binding tag and enhanced western blotting, we were able to detect LC₂₀ phosphorylation using as little as 5 pg (250 amol) of isolated LC₂₀. Phosphorylated and unphosphorylated LC₂₀ were detected in single isolated afferent arterioles and LC₂₀ phosphorylation levels could be accurately quantified in pooled samples of three arterioles (< 300 cells). The phosphorylation level of LC₂₀ in the afferent arteriole was 6.8 ± 1.7% under basal conditions and increased to 34.7 ± 5.1% and 44.6 ± 6.6% in response to 30 mM KCl and 10^-8 M angiotensin II, respectively. The application of this technique will enable investigations of the different determinants of LC₂₀ phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC₂₀ phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.