We have characterized the cis-control signals in one of the two promoters of the developmentally regulated rat insulin-like growth factor II gene (rIGF-II) by a combination of in-vivo transient expression, in-vitro transcription, footprinting, gel band-shifting and methylation-interference experiments, using a series of deletion mutant templates. Our results indicate that this simple (minimal) promoter (P2) consists of no more than 128 base-pairs, which include an ATA box and four proximal upstream GC boxes binding the general transcription factor Sp1. Three of the latter sites deviate from the known Sp1 consensus recognition sequence. The two types of cis-acting regulatory signals (GC/ATA motif) of the P2 promoter are inter-dependent and sufficient for transcription. A model for the operation of this type of minimal promoter is discussed. S1 nuclease-hypersensitive sites, localized by in-vitro mapping to the region of the P2 Sp1-binding sites, are also present in vivo and correlate with the transcriptional state of chromatin in the rIGF-II locus. We show that recognition sites for Sp1 binding are a subset of sequences that exhibit hypersensitivity to S1.