Osteopetrosis in ia rats is characterized by excessive skeletal mass and reduced bone resorption. The skeletal defects can be corrected by the transfer of mononuclear spleen cells from normal littermates. These studies suggest that osteopetrotic mutants may also have defective immune functions. The op, osteopetrotic, rat demonstrates early thymic atrophy and immune function which decreases with age. Several studies have shown significantly reduced responses to T and B cell mitogens by spleen cells from osteopetrotic mutant mice. The problem with these latter studies is that different populations of cells have been compared in mutants and normal littermates because the spleen is a focus of extramedullary hemopoiesis in osteopetrotic animals. To circumvent this problem, the Ficoll-Hypaque, mononuclear isolate of spleen and mesenteric lymph node from 5-week-old ia and normal littermates were compared. Under appropriate culture conditions the cells were exposed to Con A, PHA, and LPS for 3 days and 3H-thymidine for the last 24 hours. In all cases, the response to optimal concentrations of the 3 mitogens was similar for ia and normal spleen and lymph node cells (ia/control ratios ranged from 0.6 to 1.2). The cellular composition of the samples tested in the mitogen assays were also evaluated by fluorescent microscopy using FITC-conjugated monoclonal antibodies directed against specific cell surface markers. The percentage of B cells, macrophages, total T cells, and helper T cells were found to be similar in the Ficoll-Hypaque isolate of ia and normal spleen and lymph nodes. Likewise, the ia mutant does not show any signs of abnormal thymic involution. These results indicate normal immune function in the ia mutant when similar populations of cells are compared.