A genomic clone for starch branching enzyme (SBE) IIb was isolated from a sorghum bacterial artificial chromosome (BAC) library. The promoter and 5' flanking sequence, the first four exons and introns as well as the last exon and the 3' untranslated region were sequenced. The tentative transcription start site of sorghum sbeIIb was mapped based on alignment with the maize sbeIIb gene. The exon-intron structure of the 5' portion of sorghum sbeIIb was similar to that of maize sbeIIb but differed from that of barley sbeIIb. Specifically, the intronic BbI element involved in the endosperm specific expression of barley sbeIIb was lacking in the sorghum gene. A cDNA clone for sorghum sbeIIb was reverse PCR amplified and found to encode an 803 amino acids long protein. The amino acid sequence of sorghum SBEIIb was compared to that of sorghum SBEI and corresponding enzymes in barley. The overall identity in amino acid sequence was 54% in the central portion of the enzymes. A major difference between the SBEII and SBEI isoforms was a 67 amino acids-long C-terminal extension in the SBEIs. The spatial and temporal expression patterns of sorghum sbeIIb was determined and compared with those of the sorghum gene for SBEI and the barley genes for SBEIIB and SBEI. All four genes exhibited a seed specific expression. However, while barley sbeIIb and sbeI transcripts were detected exclusively in the endosperm, the sorghum genes were expressed also in the embryo. The activity of sorghum sbeIIb and sbeI exhibited a late onset, with a peak of transcription at around 22 days after pollination. This is similar to the pattern of barley sbeI but different from that of barley sbeIIb, which showed a peak of transcription at 12 days after pollination.