The expression of thrombospondin 1 (TSP 1), a member of the TSP gene family, is rapidly induced by growth factors. We tested the ability of human TSP 1-chloramphenicol acetyltransferase constructs to respond to serum in stably transfected NIH-3T3 cells. Two transcriptional elements in the TSP 1 promoter, a distal element at -1280 and a proximal element at -65, were required for the response of the human TSP 1 gene to serum. The distal element contains the 5'-CC(A + T)6GG-3' consensus sequence characteristic of a serum-response element (SRE). Deletions or mutations in this element reduced the serum response of the TSP 1 gene by 80-90%. In gel-shift assays, the -1280 element and the c-fos SRE cross-competed, whereas their functional and binding mutants did not. The proximal element contains the sequence 5'-GGCCAATGGG-3', which closely resembles the consensus binding motif for the CCAAT-binding factor NF-Y (CBF, CP1, alpha CP1). Deletions or mutations in this element also reduced the serum response by 80-90%. Methylation interference analysis of the -65 region identified a pattern of contacts with nuclear factors resembling that for NF-Y, and an NF-Y-binding site and the proximal TSP 1 element cross-competed in gel-shift assays, whereas their binding mutants did not. Finally, an abbreviated TSP 1 promoter/5'-flank, containing the SRE- and NF-Y-binding sites, mediated a serum response that was close in magnitude to that of the parent promoter. We conclude that the serum response of the human TSP 1 gene requires the coordinated function of an SRE- and NF-Y-binding site.