An intrasplenic injection (i.s.) of BALB/c bone marrow cells induces a higher survival rate than an intravenous injection (i.v.) in irradiated C3H/He recipients. Coculture experiments revealed the presence of alloantigen-specific and nonspecific suppressor cells in the spleens of mice injected i.s. and i.v. Suppressor activity decreased 50-60 days after bone marrow transplantation in i.v. chimeras, while there was no decrease in i.s. chimeras. In vitro suppressor activity was correlated with in vivo activity. Histopathological changes in the liver were examined. A total of 28% of the i.v. chimeras showed severe changes compared with 6% of the i.s. chimeras. The spleen indices of i.v. and i.s. chimeras were compared. Although there was no statistically significant difference between the spleen indices of i.v. chimeras and those of i.s. chimeras, spleen indices of i.v. chimeras tended to be higher than those of i.s. chimeras. These results show that suppressor cells in i.s. chimeras appear to inhibit graft-versus-host reactions more efficiently. Furthermore, an adoptive transfer assay showed that suppressor cells detected in i.s. chimeras were effective in vivo. We therefore suggest that suppressor cells detected in vitro correlate with in vivo activity and may play some role in the induction and maintenance of transplantation tolerance.