In this study, we demonstrate that the long terminal repeats (LTRs) of a murine retrovirus can activate expression of heterologous gene coding sequences from which a functional promoter region has been deleted. Recombinant plasmid clones were obtained that contained both cloned fragments of Friend spleen locus-forming virus (SFFV) DNA and the herpes simplex virus (HSV) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The effects of the LTR on tk expression were determined by constructing clones containing tk coding sequences with or without 5' sequences necessary for the initiation of transcription, inserted either 200 or 1200 base pairs downstream from the SFFV 5' LTR. The expression of the HSV TK protein by these clones was tested by gene transfer of the cloned into TK- mouse cells and assay of TK enzyme activity in TK+ transformants. These experiments demonstrate that: (i) the SFFV 5' LTR activates expression of tk coding sequences when these sequences are inserted 200 base pairs downstream from, and in the same orientation as, the LTR; (ii) tk is not activated when placed 1200 base pairs downstream from, and in the same orientation as, the LTR or when tk is inserted in either site in the opposite orientation as the LTR; (iii) the SFFV 5' LTR does not interfere with in vivo expression of tk when it is flanked by homologous 5' promoter sequences. The implication of these observations for retrovirus oncogenesis and animal cell genetics is discussed.