Nitric oxide (NO) may exert direct effects on actin-myosin cross-bridge cycling by modulating critical thiols on the myosin head. In the present study, the effects of the NO donor sodium nitroprusside (SNP; 100 microM to 10 mM) on mechanical properties and actomyosin adenosinetriphosphatase (ATPase) activity of single permeabilized muscle fibers from the rabbit psoas muscle were determined. The effects of N-ethylmaleimide (NEM; 5-250 microM), a thiol-specific alkylating reagent, on mechanical properties of single fibers were also evaluated. Both NEM (>/=25 microM) and SNP (>/=1 mM) significantly inhibited isometric force and actomyosin ATPase activity. The unloaded shortening velocity of SNP-treated single fibers was decreased, but to a lesser extent, suggesting that SNP effects on isometric force and actomyosin ATPase were largely due to decreased cross-bridge recruitment. The calcium sensitivity of SNP-treated single fibers was also decreased. The effects of SNP, but not NEM, on force and actomyosin ATPase activity were reversed by treatment with 10 mM DL-dithiothreitol, a thiol-reducing agent. We conclude that the NO donor SNP inhibits contractile function caused by reversible oxidation of contractile protein thiols.