The early promoter (P4) of the autonomous parvovirus minute virus of mice (prototype strain) directs the expression of the transcription unit coding for the nonstructural proteins NS1 and NS2. Although proximal promoter elements (GC and TATA boxes) are essential for P4 activity in vivo, additional upstream sequences appear to be required for optimal transcription. Therefore, associations of proteins with the upstream regulatory region of promoter P4 were studied in the rat fibroblast cell line FREJ4 by gel retardation and in vitro as well as in vivo footprinting assays. This led to the identification of at least four distinct upstream elements that interacted with cellular proteins. The functionality of these elements was supported by the reduced level of gene expression driven by corresponding linker-substitutive mutants of promoter P4.