To analyze transcriptional control regions of Drosophila melanogaster housekeeping genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Upstream of DmRP140 the genomic region harbors a gene which is transcribed in the opposite direction (DmRP140up). By determination of the transcription start sites of both genes we found a short non-transcribed intergenic region of 220 bp. Functional analysis of various promoter reportergene constructs by transient transfection of cultured cells revealed that sequences important for transcription of DmRP140 are located in the untranslated leader of the upstream gene. The onset of DmRP140 transcription during embryonic development was studied in transgenic flies using beta-galactosidase as reportergene. To distinguish between the maternally provided DmRP140 transcripts and the embryonically transcribed RNA the offspring of nontransformed females and male transformants was examined. The development of a sensitive detection assay based on a chemiluminescent substrate for beta-galactosidase allowed us to determine the onset of DmRP140 transcription to between 8-10 h after oviposition. Thus, DmRP140 transcription does not start following the transcriptional transition period between 2-3 h of development but occurs much later in embryogenesis coinciding with decreasing DNA synthesis and cell division rates.