The c-kit proto-oncogene is expressed in several tissues during development. To understand the mechanisms controlling the expression of this gene, we characterized the human c-kit promoter. Expression is controlled transcriptionally. The 5'-flanking DNA was used to make promoter deletion-reporter constructs that were tested in cells that were either positive or negative for endogenous c-Kit. The results demonstrate that DNA, to at least position -4100, directs transcription well in both positive and negative cells. Addition of DNA from position -4100 to -5500 causes a reduction in expression to near-basal levels in c-Kit-negative cells but has little effect in c-Kit-positive cells. The DNA from -4100 to -5500 was tested for repressor function. It inhibits transcription from some heterologous promoters in c-Kit-negative cells. Likewise, this segment inhibits transcription from the homologous proximal promoter in a cell-specific manner, but the entire promoter is necessary for complete repression in c-Kit-negative cells. Two Myb binding motifs were also identified, and their role in regulating transcription was examined by mutation and functional testing. One, MYB1, acts as a partial repressor, whereas the other, MYB2, is a positive element that appears essential for expression. Binding proteins to both sites were characterized by several methods. MYB1 binds and responds functionally to c-Myb, but MYB2 does not. The results of these studies indicate that the regulation of c-kit transcription is complex, involving interactions among several activators and repressors.