The 5'-upstream region of the rat phospholipase C-beta 3 gene (PLC-beta 3) has been cloned and characterized. Sequence analysis of the 5'-upstream region showed that it contains a GC-rich region (-166 to +1: 79%) and multiple binding sites for the transcription factors Sp1, AP-1 and AP-2, but does not contain a canonical TATA box. Primer extension analysis of total RNA isolated from rat glial cell C6Bul revealed that single transcription start point (tsp) is located at an initiator (Inr) element similar to that found in the HIV promoter. Gel mobility shift and competitive mobility shift assays indicated that this Inr element forms a DNA-protein complex with the HIV Inr-binding protein, LBP-1/CP2 or a homologue. In order to localize functional elements of the 5'-upstream region of the rat PLC-beta 3 gene, 5'-deletion fragments were cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. Transient transfection analyses of the 5'-deletion mutants identified a crucial promoter element located at -128 to -14. Supershift mobility assays, site-directed mutagenesis and DNase I footprints indicated that Sp1 binds to three GC boxes within the sequence between -128 and -14 of the PLC-beta 3 promoter. Transient transfection analyses of promoter constructs containing site-specific mutation(s) of these three GC boxes demonstrated that two GC boxes, located proximal to the tsp, are important elements for normal promoter activity.