The subgenomic mRNAs (sg mRNA) of porcine reproductive and respiratory syndrome virus (PRRSV) were characterized. The number of sg mRNAs, which form a 3'-coterminal nested set in PRRSV-infected cells, varied from six to seven among PRRSV isolates with differing virulence. The additional species of sg mRNA in some isolates of PRRSV was designated as sg mRNA 3-1. The leader-mRNA junction sequences of sg mRNAs 3, 3-1 and 4 were found to contain a similar six nucleotide sequence motif, U(G)UA(G/C)ACC. By comparing the 5'-terminal sequence of sg mRNA 3-1 with the genomic sequence of two isolates, ISU79 and ISU1894, it was found that a point mutation, from U in isolate ISU1894 to C in isolate ISU79, led to the acquisition of a new leader-mRNA junction sequence (UUGACC) in isolate ISU79, and therefore an additional species of sg mRNA 3-1. A small ORF (3-1) was identified at the 5' end of sg mRNA 3-1.