The concentrations of platelet-activating factor (PAF) that possesses the ability to stimulate myometrial contraction are partially regulated by intracellular type of platelet-activating factor acetylhydrolase (PAF-AH) in many tissues. Tissue cytosol contains at least two intracellular PAF-AH, isoforms I and II. To examine the relationship between the activity and isoforms of intracellular PAF-AH in human uterine myometrium and myoma, we assayed the PAF-AH activity and identified the PAF-AH isoforms I and II by Western blot analysis. The intense bands of the α2 and β subunits of PAF-AH isoform I were detected in nonpregnant uterus; however, the specific bands of the α1 subunit of PAF-AH isoform I and the PAF-AH isoform II were extremely weak. The levels of the α2 and β subunits and PAF-AH activity in pregnant uterus (37–39 wk gestation) were significantly lower than those in nonpregnant uterus. On the other hand, the level of β subunit and the PAF-AH activity in myoma were significantly higher than those in nonpregnant uterus. No significant difference was found in the expression of the PAF-AH isoform II among three tissues. These results indicate that the change in the PAF-AH activity observed in pregnant uterus and myoma are due to the lower or higher protein expression of the PAF-AH isoform I, especially the α2 and/or β subunits. The decrease of the uterine PAF-AH activity in the late stage of pregnancy may facilitate the action of PAF to stimulate myometrial contraction.