To identify features of the human thrombospondin 2 gene (THBS2) important for regulation of expression, the sequences of 5 kb of the promoter/5′ flank and 3 kb of transcribed and intronic DNA were determined. Two repetitive sequences were found: an MLT1c element located 2.2 kb 5′ of exon 1 and, further 5′, 1.8 kb of a Tigger1 element. Putative transcription factor binding sites that might be significant for THBS2 regulation included p53, NF-kB, Sp1, Myc-CF1, NF-Y, CF1, AP1, and GATA sites. Alignment of the promoter/5′ flank sequence with the mouse Thbs2 promoter revealed 78% identity for a 450 bp region immediately upstream from the mouse transcription start site. No significant homology was detected between the human thrombospondin 2 and thrombospondin 1 promoters. Comparison of the THBS2 genomic and cDNA sequences revealed that, in contrast to Thbs2, exon 1 is divided into exons 1A and 1B by a small (93 bp) intron. The transcription start site was investigated by a PCR procedure and by 5′ RACE, and yielded a size for exon 1A of at least 186 bp. Tissue-specific differences in transcription start sites were found, with transcript lengths in the order: fetal lung>adult lung>fetal brain. These results suggest that tissue-specific differences in expression of the THBS2 gene may be determined, in part, by selection of the transcription start site and resulting differences in the 5′ untranslated region.