A flow microcalorimetric technique was used for the first time to evaluate the effect of myosin phosphorylation on actomyosin ATPase activity. Using an actomyosin system containing myosin light-chain kinase and phosphatase, myosin phosphorylation affects the actomyosin ATPase activity positively or negatively depending upon the molar myosin:actin ratio used in the assay. Our results show that under conditions near physiological ones the phosphorylation of myosin light chains decreases the actomyosin ATPase activity.