The nuclear transcription factor c-Myb, which is highly expressed in hematopoietic cells, has been shown to be functional in NIH 3T3 cells: cells that do not possess detectable levels of c-Myb. To identify endogenous target genes of c-Myb in fibroblasts, RNA isolated from NIH 3T3 cells stably transfected with a full-length or a dominant negative c-myb construct (GREMyb and GREMEn, respectively) was subjected to differential display analysis. 5'-Rapid amplification of cDNA ends of a selected band, sequencing, and a nucleotide homology search led to the identification of thrombospondin 2 (TSP 2) as the gene product repressed in GREMyb and induced in GREMEn cells. The pattern of TSP 2 expression during the cell cycle was consistent with c-myb-dependent regulation. The possibility that the identified transcript was TSP 1, a homologous product known to be repressed by v-Src, c-Jun, and v-Myc, was ruled out by using a TSP 2-specific DNA probe and by showing a distinct pattern of regulation of TSP 1 and TSP 2 expression. Nuclear run-on and TSP 2 promoter-reporter (chloramphenicol acetyltransferase) assays showed similar transcriptional levels in GREMyb and NIH 3T3 cells. However, mRNA stability studies showed a much shorter TSP 2 mRNA half-life in GREMyb compared with wild type NIH 3T3 cells, suggesting that c-myb affects TSP 2 expression via a post-transcriptional mechanism. The implications of a protooncogene-mediated suppression of TSP expression are discussed.