Previous studies have shown that prolonged estrogen treatment of mice markedly reduces their natural killer (NK) cell activity. Our experiments demonstrate that splenocytes from (C57BL/6 X C3H/He) F1 mice treated with 17 beta-estradiol are suppressive for the NK activity of splenocytes from untreated mice when the two cell populations are mixed during cytotoxicity assays. The suppressor activity is resistant to treatment with anti-Thy-1.2 or anti-la reagent plus complement, can be generated in neonatally thymectomized mice, and is present in plastic-adherent as well as nonadherent cell populations. Treatment with a NK-reactive antiserum, either anti-asialo-GM-1 or anti-NK-1.2, has no effect on the suppressor activity. Administration of the interferon inducer polyinosinic-polycytidylic acid to mice treated with estrogen results in moderate restoration of NK activity but has no effect on the suppressor activity. These data suggest that generation of a Thy-1-negative/la-negative suppressor cell population is, at least in part, responsible for the reduced levels of NK activity in estrogen-treated mice.