Soybean contains a multigene family which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCss). A member of this gene family, SRS4, has been isolated from a soybean genomic DNA library. Its nucleotide sequence has been determined and compared to the sequence of SRS1, a previously characterized RuBPCss gene from soybean. Relevant regulatory sequences such as the PuPuCCAAT boxes, TATA box, the actual start of transcription and poly A addition sites are conserved between the two genes. Using a gene specific synthetic probe to the 3′ flanking region the steady state mRNA levels of SRS4, like SRS1, are shown to be very high in light grown soybean seedlings and low in seedlings grown in darkness. SRS1 and SRS4 are very closely related, the three exons being 96%, 93% and 96.5% homologous in nucleotide sequence. The polypeptide sequences are nearly identical with only one amino acid change in each of the three exons encoding the 178 amino acid precursor polypeptide. The two introns are about 75% homologous and the flanking regions are more than 85% homologous (700 base pairs on the 5′ end and 300 base pairs on the 3′ end). Furthermore, hybridization studies between lambda clones containing the SRS1 and SRS4 genes reveal that a region of strong homology extends at least 4 kb on the 5′ end and about 1.1 kb on the 3′ end. We propose that these two genes may be alloalleles or homeologous alleles. This proposal is consistent with soybean having an allotetraploid origin, and would imply that the divergence of two ancient Papilionoidae species gave rise to these two genes.