The GA-binding protein (GABP), a heterodimeric transcription factor with widespread tissue distribution, has been found to be a strong positive regulator of several ribosomal protein (rp)-encoding genes. In such genes, e.g. the mouse rpL30 gene, the GABP-binding sites are located 40-80 base pairs upstream of the transcriptional start point. Potential GABP-binding sites are present in the promoters of numerous other rp genes, not only in upstream regions, but also in the immediate vicinity of the transcriptional start point. The mouse rpS16 gene is an example of the latter type. We demonstrate here that GABP binds to the rpS16 initiation region, and in so doing down-regulates rpS16 transcription both in vivo and in vitro. Supplementation of cell-free extracts with GABP inhibits transcription on rpS16 templates while concomitantly stimulating transcription on rpL30 templates. The repressive and stimulatory effects, which were proportional to the amount of GABP added, required both the GABP alpha subunit and either a beta1 or beta2 subunit. Mutations of the rpS16 GABP-binding sites that abolish binding increased rpS16 promoter activity in vivo and in vitro, whereas mutations that strengthen GABP binding caused a reduction in promoter activity. The binding of GABP to the rpS16 initiation region does not significantly affect the positioning of the transcriptional start points. Taken together with earlier studies, these new findings indicate that GABP can have a dual role as repressor or activator of rp gene transcription.