Galphai2-deficient mice, which spontaneously develop colitis, have previously been reported to have an increased frequency of mature, single positive thymocytes compared to wild-type mice. In this study we further characterized the intrathymic changes in these mice before and during overt colitis. Even before the onset of colitis, Galphai2(-/-) thymi weighed less and contained fewer thymocytes, and this was exacerbated with colitis development. Whereas precolitic Galphai2(-/-) mice had unchanged thymocyte density compared to Galphai2(+/-) mice of the same age, this was significantly decreased in mice with colitis. Thymic atrophy in Galphai2(-/-) mice involved mainly the cortex. Using a five-stage phenotypic characterization of thymocyte maturation based on expression of CD4, CD8, TCRalphabeta, CD69 and CD62L, we found that both precolitic and colitic Galphai2(-/-) mice had significantly increased frequencies of mature single-positive CD4(+) and CD8(+) medullary thymocytes, and significantly reduced frequencies and total numbers of immature CD4(+) CD8(+) double-positive thymocytes compared to Galphai2(+/-) mice. Furthermore, cortical and transitional precolitic Galphai2(-/-) thymocytes showed significantly reduced chemotactic migration towards CXCL12, and a trend towards reduced migration to CCL25, compared to wild-type thymocytes, a feature even more pronounced in colitic mice. This impaired chemotactic migration of Galphai2(-/-) thymocytes could not be reversed by increased chemokine concentrations. Galphai2(-/-) thymocytes also showed reduced expression of the CCL25 receptor CCR9, but not CXCR4, the receptor, for CXCL12. Finally, wild-type colonic lamina propria lymphocytes migrated in response to CXCL12, but not CCL25 and, as with thymocytes, the chemokine responsiveness was significantly reduced in Galphai2(-/-) mucosal lymphocytes.