Regions of open reading frame (ORF) from the genome of the autonomous parvovirus minute virus of mice (MVM) were cloned into a procaryotic expression vector, and bacterial fusion proteins containing MVM-specific amino acid sequences were isolated. Antibodies raised against these proteins were used to immunoprecipitate viral proteins synthesized in vitro in a rabbit reticulocyte lysate translation system programmed with mRNA isolated from cells infected with MVM and a number of different parvoviruses. These studies demonstrated that: the 83-kilodalton nonstructural protein NS-1 and the 25-kilodalton nonstructural protein NS-2 have a common amino-terminal sequence which is encoded by the single ORF located between nucleotides 225 and 534 in the viral genome; the ORF located between nucleotides 1110 and 1638 is only expressed in the NS-1 protein; and the sequence encoded in a small alternative ORF between nucleotides 2075 and 2291 is expressed exclusively in NS-2. These data confirm that NS-1 is the product of the 4.8-kilobase R1 viral transcript and demonstrate that NS-2 is synthesized from the 3.3-kilobase R2 transcript which arises from the left-hand promoter at map unit 4 on the viral genome. Antibodies against the MVM fusion proteins also cross-reacted with similar proteins encoded by the viruses H-1 and LuIII, but although antibodies against the carboxy-terminal half of NS-1 cross-reacted with a similar protein in CPV, we were unable to demonstrate an NS-2 protein encoded by this virus.