The orphan nuclear receptor, retinoid acid-related orphan receptor (ROR)α, is essential for the development of cerebellar Purkinje cells and bone tissue. RORα may also play a critical role in lymphocyte development and function because staggerer mice, a natural mutant strain with a disrupted expression of RORα, have reduced thymic and splenic cellularity. In this report, we analyzed the of RORα in lymphocyte development by examining lymphoid compartments in RORα-/- mice and Rag-2-/- mice reconstituted with RORα-/- bone marrow. We found that T and B cell development was severely defective in RORα-/- mice, but not in Rag-2-/-/RORα-/- chimeric mice. We also analyzed cellular and humoral immune responses in Rag-2-/-/ RORα-/- chimeric mice. Our results show that serum IgG levels were elevated in Rag-2-/-/RORα-/- chimeric mice after immunization with a T-dependent Ag compared with control chimeras. IFN-γ production by RORα-/- CD8+ T cells after TCR stimulation was also increased. Furthermore, RORα-/- mast cells and macrophages produced an increased amount of INF-α and IL-6 upon activation. These results indicate that RORα indirectly regulates lymphocyte development by providing an appropriate microenvironment and controls immune responses by negatively regulating cytokine production in innate immune cells and lymphocytes.