The kinetics of myosin light chain (LC20) phosphorylation has been studied during in vitro contraction and relaxation of rat uteri. Phosphorylation preceded contraction and continued during tetanus induced by KCl; the degree of phosphorylation was proportional to the percentage of contraction. On the other hand, during relaxation induced by isoproterenol, the level of phosphorylation did not change in the seconds following relaxation. A rapid change in cAMP did not appear to trigger a rapid change in LC20 phosphorylation. In cyclic rats, progesterone decreased the extent of LC20 phosphorylation: 50%-60% of the LC20 was phosphorylated in untreated animals or estrogen treated animals. This value fell to 30% after progesterone treatment of cyclic rats. In ovariectomized rats, steroid hormones did not affect the phosphorylation reaction. Under the same conditions, the level of cAMP-dependent protein kinases did not change during the cycle, or after estradiol-treatment, but in cyclic rats it doubled after progesterone treatment. Hence, the decrease in the level of LC20 phosphorylation observed in cyclic rats treated by progesterone could be due to a decrease in myosin light chain kinase (MLCK) activity, resulting from a higher proportion of the phosphorylated form of this enzyme. The concomitant increase in the proportion of activatable cAMP-dependent protein kinases could favor the maintenance of a higher level of phosphorylated MLCK for longer periods of time.