Estriol (E(3)), an endogenous estrogen predominantly produced during human pregnancy, has been suggested to play an important role in modulating the immune system function during pregnancy. The present study sought to investigate the ability of E(3) to alter splenocyte functions in non-immunized naïve BALB/c female mice and also in mice injected with complete Freund's adjuvant (CFA), and the effect of E(3) was compared with that of 17β-estradiol (E(2)). When mice were injected with CFA, their spleen weight index (i.e., wet organ wet/whole body weight) was increased by ~ 300%, but co-administration of E(3) almost completely suppressed splenomegaly. E(3) also reduced cytokine production and reduced ERK and p38 activation in both splenocytes and peritoneal exudate cells from CFA-treated animals. In comparison, while E(2) had a similar but slightly weaker effect than E(3) in reducing splenomegaly, it had a rather different effect from E(3) on cytokine production and ERK activation in splenocytes and peritoneal exudate cells from CFA-treated mice. Under naïve immunological conditions, E(3) and E(2) had very similar effects on splenocyte functions. Both of them transiently increased the percentages of splenic CD4(+) and CD8(+) cells. They also increased the proliferation of splenocytes ex vivo, and stimulated production of interferon-γ and interleukin-2. Altogether, these data show that E(3) and E(2) have different effects on splenocyte functions when the animals are under experimentally induced inflammatory conditions.