Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.