CD45, a receptor-type protein tyrosine phosphatase involved in lymphocyte activation, consists of five isoforms generated by alternative mRNA splicing. The pattern of CD45 splicing depends upon cell type, state of activation, and thymic development. We previously showed that mouse B and thymocyte cell lines transfected with a human CD45 minigene construct appropriately splice the alternative exons, demonstrating the presence of trans-acting tissue-specific splicing factors. To study the regulation of CD45 alternative splicing, cells having different splicing patterns were transiently fused, and mRNA was analyzed using reverse transcription-polymerase chain reaction. Human B cells, normally producing only the largest CD45 mRNAs, could splice out the alternative exons after fusion with mouse thymocytes. In contrast, the splicing pattern of human T cell lines (smaller CD45 isoforms) was unaltered by fusion with mouse B cells. This suggests that cells expressing the smallest CD45 isoform contain negatively acting trans-factor(s) that allow the alternative exons to be skipped, and that the full length isoform containing all three alternative exons is the default pattern of splicing. In agreement, incubation of thymocytes or T cell subsets with cycloheximide increased mRNA corresponding to the larger isoforms. CD45 isoform expression can now be interpreted in terms of the presence or absence of negative regulatory trans-acting splicing factors.