Deletional analysis within the long terminal repeat (LTR) of Moloney murine leukemia virus (M-MuLV) was performed. By molecular cloning, deletions were made in the vicinity of the XbaI site at -150 base pairs (bp) in the U3 region, between the tandemly repeated enhancers and the TATA box. The effects of the deletions on LTR function were measured in two ways. First, deleted LTRs were fused to the bacterial chloramphenicol acetyltransferase gene and used in transient expression assays. Second, infectious M-MuLVs were generated by transfection of M-MuLV proviruses containing the deleted LTRs, and the relative infectivity of the mutant viruses was assessed by XC-syncytial assay. Most of the deleted LTRs examined showed relatively high promoter activity in the transient chloramphenicol acetyltransferase assays, with values ranging from 20 to 50% of the wild-type M-MuLV LTR. Thus, the sequences between the enhancers and the TATA box were not absolutely required for transient expression. However, infectivity of viruses carrying the same deleted LTRs showed more pronounced effects. Deletion of sequences from -195 to -174 bp reduced infectivity 20-to 100-fold. Deletion of sequences within the region from -174 to -122 bp did not affect infectivity, indicating that this region is dispensable. On the other hand, deletion of sequences from -150 to -40 bp reduced infectivity from 5 to 6 logs, although the magnitude of the reduction partly may have reflected threshold envelope protein requirements for positive XC assays. The reduced infectivity did not appear to result from a failure of proviral DNA synthesis or integration by the mutant. Thus, the infectivity measurements identified three functional domains in the region between the enhancers and the TATA box.Retroviruses generate viral DNA with long terminal repeats (LTRs) during reverse transcription (reviewed in references 21 and 22). The LTRs are essential for viral replication and expression because they contain the signals for viral DNA integration, transcription initiation, and RNA cleavage and polyadenylation. In particular, the U3 portion of the LTR contains canonical eucaryotic promoter signals (TATA and CCAAT homologies at approximately -30 and -80 base pairs [bp], respectively) as well as transcriptional enhancers ( . approximately -340 to -180 bp) (10,20). As such, the retroviral LTR is an excellent model system for studying normal eucaryotic gene expression.In the experiments reported here, sequences in the U3 portion of the Moloney murine leukemia virus (M-MuLV) LTR necessary for LTR function were investigated by deletional analysis. We previously demonstrated the requirement for the enhancers (12), so emphasis was placed on the sequences between the enhancers and the TATA box. The activities of the deleted LTRs in transient expression assays were assessed, similar to experiments reported by others (8, 10). In addition, we extended the analyses by generating infectious M-MuLVs containing the deleted LTRs.