The nucleocapsid protein of the European genotype of porcine reproductive and respiratory syndrome virus (type 1, PRRSV-1) exhibited extensive size polymorphism (124-130 amino acids), correlating with phylogenetic grouping of ORF7 as well as ORF5 nucleotide sequences, thereby validating ORF7 size as an independent PRRSV-1 subtype marker. Based on new sequence information from the Russian Federation, we propose division of European genotype PRRSV-1 into 3 subtypes: a pan-European subtype 1 and East European subtypes 2 and 3, with nucleocapsid protein sizes of 128, 125 and 124 amino acids, respectively. The genetic differences between European genotype PRRSV subtypes affected diagnostic RT-PCR primer binding sites. Using Escherichia coli-expressed ORF7 protein, we confirmed that even the relatively closely related PRRSV subtypes 2 and 3 were antigenically different. Finally, the isoelectric point (pI) correlated with the nucleocapsid protein size for European genotype PRRSV subtypes, suggesting subtype-specific compensatory structural changes associated with subtype-specific ORF7 sizes. Thus, the new ORF7-based subtype division of PRRSV-1 proposed here is biologically meaningful and practically relevant.