Alternative splicing generates various Ly-5 glycoprotein isoforms of the cell surface that typify different cell lineages and stages of hematopoietic differentiation in the mouse; exons 4-6 are incorporated to generate a B-cell isoform (B220) and excluded from a T-cell isoform (T200), the other coding exons (3 and 7-33) being shared. As a first step to understanding the mechanisms regulating Ly-5 alternative splicing, and thus determining Ly-5 isoforms, a minigene representing exons 3-7 was transfected into Ly-5-expressor T cells and B cells and into nonexpressor L cells for comparison of splicing patterns. We conclude that all the information required for faithful splice-site selection according to cell type is contained within the resulting pre-mRNA. The splicing pattern manifested by nonexpressor L cells may represent a default and nonregulated type. We postulate trans-acting factor(s) to account for the selection of appropriate exons, and we provide support for this interpretation from analysis of fused hybrid T-B cells, which exhibited B-cell specific Ly-5 transcripts. Splicing patterns were well conserved despite substantial disruption of constructs. However, extensive deletion analyses suggested that cis sequences flanking and within exon 6 affect the exclusion of that exon in T cells.