We have obtained correct transcripts from the mouse immunoglobulin lambda I light chain gene on transfection into human HeLa cells. Linkage to simian virus 40 (SV40) DNA containing a transcriptional "enhancer" element was required to raise lambda-chain gene transcription to a detectable level in our transient-expression assay. The transcripts had the same 5' end as authentic lambda I mRNA when the SV40 enhancer element was 150 base pairs upstream from the cap site. The situation was different when the lambda-chain gene promoter was separated from the SV40 sequences by more than 1 kilobase pair of spacer DNA; then, lambda-chain gene transcripts were not correctly initiated in human HeLa, monkey CV-1, and mouse 3T6 cells. In this respect, the lambda-chain gene behaves differently from the rabbit beta-globin gene, which can be activated by the SV40 enhancer over distances of several kilobases.