The G-C specific DNA binding drug mithramycin selectively inhibits expression of the c-myc gene in a number of cell types. We have tested the ability of this agent to inhibit the expression of a transfected human c-myc gene in a murine fibroblast cell line. Expression of c-myc is inhibited in the first 24 hours of mithramycin exposure (in contrast to actin gene expression, which is unaffected). Nuclear runon transcription of c-myc by nuclei isolated from mithramycin treated cells is decreased, indicating inhibition of transcription initiation. However, treatment of isolated nuclei with mithramycin also results in decreased c-myc transcription. Thus, inhibition of c-myc expression by mithramycin in these cells appears to occur at the transcriptional level and is most likely mediated at both the transcription initiation and elongation level. This suggests that mithramycin selectively interacts with the G-C rich c-myc promoter, preventing formation of the c-myc transcription initiation complex.