We report the use of a HeLa whole cell extract (WCE) runoff transcription system for the study of cis- and trans-acting elements, participating in the regulation of transcription initiation from the P38 promoter of the parvovirus minute virus of mice (MVM). Our initial studies with HeLa WCE indicated that transcription from the P38 promoter is very inefficient, compared with transcription from the P4 promoter. Supplementation of the HeLa WCE with WCE prepared from uninfected Ehrlich ascites cells enhanced transcription from the P38 promoter twofold, indicating a role for a cellular factor in transcription from the P38 promoter. Furthermore, supplementation with WCE prepared from MVM-infected Ehrlich ascites cells enhanced transcription from the P38 promoter about sixfold, indicating a role for a virally encoded or induced factor. Analyses of runoffs produced by transcription of DNA templates digested with various restriction enzymes defined a downstream promoter element (DPE) necessary for efficient transcription initiation from the P38 promoter. This element resides 282 to 647 base pairs 3' to the transcription initiation site, between the NarI site and the HindIII site (2287 to 2652, MVM numbering system). The virally encoded NS1 protein was shown by DNA precipitation to bind directly or indirectly through a cellular factor to the DPE. This interaction is suggested to be involved in the up regulation of the P38 promoter of MVM. Finally, with a DNase I protection assay performed on a fragment containing the DPE, we estimated the sequence involved in the binding of a factor present in uninfected and infected extracts. The correlation between the binding and transcription activation is discussed.