We have used protoplasts derived from a maize (Black Mexican Sweet) suspension culture to study the replication of maize streak virus (MSV) wild-type (wt) and mutant DNA genomes. Following inoculation with plasmids containing multimeric copies of MSV, both single-stranded (ss) and double-stranded MSV DNA forms were produced, in proportions relative to those seen in infected plants. The immunodetection of coat protein (PV2) and geminate particles confirmed that the protoplasts were able to support the entire multiplication cycle of MSV and therefore were suitable for the study of MSV gene function. Inoculation of protoplasts with V1 gene mutants which are unable to produce systemic infections in plants resulted in DNA replication and encapsidation indistinguishable from that obtained with wt constructs implicating the protein in virus movement. Computer analysis of the V1 protein (PV1) indicated a potential trans-membrane or membrane-embedded domain. Following inoculation of protoplasts with V2 mutants, ssDNA was not detected; the inability of these mutants to spread in plants may be due to a lack of ssDNA, PV2, or a combination of the two. The requirements for systemic infection of MSV are discussed.