Thrombospondin 3 (TSP3) is a secreted, pentameric glycoprotein whose regulation of expression and function are not well understood. Mouse Thbs3 is located just downstream from the divergently transcribed metaxin gene (Mtx), which encodes an outer mitochondrial membrane import protein. Although Thbs3 and Mtx share a common promoter region, previous studies showed that Mtx is regulated by proximal elements that had little effect on Thbs3 expression. In this study, transient transfection of rat chondrosarcoma cells and NIH-3T3 fibroblasts demonstrated that Thbs3 is regulated in a cell type-specific manner by a position- and orientation-independent far upstream enhancer located within intron 6 of Mtx. Despite its greater proximity to the transcription start site of Mtx, the Thbs3 enhancer did not have a significant effect on Mtx expression. Two DNA-protein complexes, which were both required for activity, were identified when nuclear extracts were assayed with a probe containing the enhancer sequence. The protein in one of these complexes was identified as Sp1, while the other DNA-protein complex remains uncharacterized. A 6-kilobase pair promoter containing the enhancer was able to direct specific expression of the E. coli lacZ gene in transgenic mice, whereas a 2-kilobase pair promoter that lacked the enhancer was inactive. Thus, despite their close proximity, the genes of the Mtx/Thbs3 gene cluster are regulated independently.