CD56+ CD16- granulated lymphocytes, termed endometrial granulated lymphocytes (eGLs), have been suggested to play a role in the maintenance of human pregnancy, although their in vivo function in both pregnant and nonpregnant endometrium remains unknown. The present study compared the cytotoxic activity of CD56+ CD16- eGLs (> 98% purity) positively selected from early and late proliferative-phase, early and late secretory-phase, and menstrual-phase endometrium with that of CD56+ CD16- eGLs purified from first-trimester decidua and CD56+ predominantly CD16+ cells from peripheral blood. From the late proliferative phase onwards, the major histocompatibility complex (MHC)-nonrestricted cytotoxic activity of eGLs was comparable between phases of the menstrual cycle. In contrast, eGLs from early proliferative-phase endometrium displayed significantly lower cytotoxic activity. With the exception of eGLs purified from early proliferative-phase endometrium, the cytotoxic activity of CD56+ CD16- eGLs purified from nonpregnant endometrium was comparable to that of CD56+ CD16- eGLs in decidua and CD56+ predominantly CD16+ cells from peripheral blood. No endogenous lymphokine-activated killer cell activity was detected in eGLs from endometrium or decidua. The present study using highly purified eGLs demonstrates that, with the exception of early proliferative-phase samples, CD56+ CD16- eGLs from nonpregnant endometrium and early pregnancy decidua have cytotoxic activity comparable to that of "classical" natural killer cells from peripheral blood.