Recombinants in which the six GC-motifs (I-VI) present in the upstream element of the SV40 early promoter region have been point mutated either individually or in pairs were used to determine the possible contribution of each GC-motif to the function of the overlapping early-early and late-early SV40 promoters. GC-motif I, and to a lesser extent, GC-motifs II and III, are critical for initiation at the early-early start sites. GC-motifs IV-VI play a subsidiary role. Mutations in GC-motifs I and II do not decrease the activity of the late-early promoter, whereas mutations in the GC-motifs III-VI have a moderate effect on it. The in vivo phenotype of the GC-motif mutants can be almost fully reproduced in vitro using a nuclear extract. DNase I protection footprinting experiments using wild-type or mutated templates and nuclear extracts indicate that each GC-motif behaves principally as an independent protein-binding site, presumably for transcription factor Sp1. The effect of changing the position of the 21-bp repeat region on initiation from the early-early and late-early start sites indicates that there is little flexibility in the position in which this upstream element can efficiently activate initiation of transcription from these start sites.