Previous in vivo experiments provided evidence of suppressive activity induced by multiple allogeneic pregnancies. The reactivity of maternal spleen cells toward paternal strain alloantigens was investigated by use of the MLR [mixed lymphocyte culture] microculture technique. A study of the kinetics of the MLR showed an early peak of reactivity (48 h culture) followed by a decline leading to a decreased reactivity by 96 h when spleen cells from allogeneically pregnant mice were compared to those of virgin or even isogeneically pregnant mice, suggesting the possible action of MLR regulatory cells. A strong suppression of a H-2k (CBA) anti-H-2a (A/J) or anti-H-2d (C57BL/Ks) MLR was observed when mitomycin-treated spleen cells from CBA mice multiparous by A/J or C57BL/Ks (but not CBA) males were added to the culture. This suppression was abolished by treating the regulatory cell population with anti-.theta. serum plus complement or replacing the 1% normal mouse serum in the medium by a proper anti-idiotypic mouse serum.