The human testis-specific lactate dehydrogenase c gene (ldh-c) shows an exceptionally large window of expression throughout pre- and postmeiotic stages of the male germ cell lineage. In order to characterize the multiple stage-specific transcription factors necessary for ldh-c expression, we previously characterized the human ldh-c core promoter. Here, we used a combination of gel retardation assays and an in vitro transcription system derived from human tissues to better define the elements that govern ldh-c transcription. Three classes of transcriptional regulators were defined by these experiments. 1) The Sp1 transcription factor is a testis-"enriched" protein that is absent from most somatic tissues and that appears to play a major role in determining ldh-c expression in the testis. Highest levels of Sp1 during spermatogenesis correlate with maxima of ldh-c expression. 2) The testis-specific cAMP response element modulator (CREM) transcription factor binds a cAMP response element (CRE)-like sequence located at position -433. This transcriptional activator might contribute to postmeiotic transcription of ldh-c. 3) Factors present in tissues negative for ldh-c expression appear to bind both the CRE-like sequence and an adjacent hormone response element. The presence of this element could be involved in regulating ldh-c through the glucocorticoid/androgen pathways at the early stages of ldh-c expression.