The VpreB genes, which encode surrogate immunoglobulin light chain molecules, are expressed as RNA almost exclusively in pre-B cells. We have investigated the transcriptional control mechanisms which are responsible for the pre-B cell-specific RNA expression of the mouse VpreB1 and VpreB2 genes. Nuclear run-on analyses demonstrate that the pre-B cell-specific expression of both VpreB genes is controlled primarily at the level of initiation of transcription. S1 nuclease protection-mapping defined two or three major start sites of transcription for the VpreB genes. To find a promoter and other potential cis-acting regulatory elements, a 700-bp fragment 5' of the transcription start sites of the VpreB1 gene was used in gene transfer experiments and found to act as a promoter in pre-B lymphocytes. Deletion experiments showed that 191 bp upstream of the most 5' transcription start site is required for the pre-B cell promoter activity. DNA sequence analysis of the 5' region of the mouse VpreB1, VpreB2 and human VpreB genes reveal that this region of approximately 200 bp is strongly conserved. This 200-bp promoter region contains several conserved nucleotide sequence motifs which may act to mediate the pre-B cell-specific transcription of the VpreB genes.