The promoter of the mouse lactate dehydrogenase c (Ldh-c) gene was assayed in an in vitro transcription system. Of the nuclear extracts isolated from several mouse tissues, only testis supported transcription from a 710 Ldh-c promoter fragment. Mutation analysis indicated that a canonical TATA box and a 30-bp palindromic sequence are both required for promoter activity. Liver nuclear extract (LN) can reduce transcriptional activity significantly when added to testis nuclear extract (TN). Deletion of the 3' palindromic sequence resulted in a low level of transcriptional activity in LN. Competition with a double-stranded synthetic palindromic oligonucleotide inhibited transcription in TN and rescued a low level of transcription in LN. Our results strongly indicate that the palindromic sequence contains both a negative element for repression in somatic tissues and a positive element for activation of the Ldh-c gene in testis.