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An alternatively spliced mRNA of pulmonary surfactant protein B (SP-B) was identified in murine lung. Sequencing analysis revealed a 69 base-pair (bp) deletion at the beginning of exon 7 of SP-B, presumably the result of an alternative splicing event. Reverse transcription-polymerase chain reaction (RT-PCR) of mouse, rat, and rabbit lung RNA revealed the existence of full-length and the 69-bp deleted short form. Ribonuclease protection assay of the SP-B messenger RNA (mRNA) demonstrated expression of both isoforms in five strains of adult and fetal mice with different genetic backgrounds, as well as in rabbit, but not in human. Splice junction sequences in exon 6 and at the exon 7 splice boundary for the two isoforms are similar, including AG doublet identity, but sequence differences do not account for species variation in isoform abundance. The abundance of the short SP-B mRNA isoform was approximately 30% of total SP-B mRNA in mouse and rabbit. Analysis of precursor SP-B protein in mouse lung suggested that the two mRNA species are expressed as stable protein isoforms.