The nucleotide sequence of the L gene of vesicular stomatitis virus, New Jersey serotype (Hazelhurst subtype), was determined. Primer extension dideoxy sequencing of genomic RNA using reverse transcriptase initiated within the adjacent G gene provided a consensus sequence of 6522 nucleotides. The G/L intergenic junction spanned 21 nucleotides and contained a pseudo transcription start signal as well as two sequences (10 and 6 nucleotides in length) which are reiterated within the L coding region. The predicted L mRNA was 6398 nucleotides long and contained a single open reading frame corresponding to an L protein encompassing 2109 amino acids with a MW of 241,546. Comparison of the amino acid sequence of this New Jersey serotype L protein to that previously reported for the L protein of the serologically and genetically distinct Indiana serotype (M. Schubert, G. G. Harmison, and E. Meier (1984). J. Virol. 51, 505-514.) revealed a high degree of functional homology. In addition, six regions (43 to 103 amino acids in length) which displayed a high percentage of identical amino acids (85 to 96%) were identified. Five of these regions were clustered within the amino-terminal half of the L protein. Two of these regions contained sequences, 41 amino acids in length, which were significantly similar to corresponding regions of the L proteins of the paramyxoviruses Sendai and Newcastle disease virus. These structurally conserved regions may correspond to functional domains of the multifunctional L protein.