The infectivity of cloned unit-length genomes of Maize streak virus (MSV) was tested using vascular puncture inoculation (VPI). VPI of kernels with plasmid DNA (pUC19) carrying a tandem repeat of the MSV genome produced 33+/-8% infection. Similar plasmids carrying the unit-length MSV genome were not infectious. If the MSV genome was released from the plasmid prior to VPI, 16+/-4% of plants became infected. Ligation of the free linear MSV genome did not increase infectivity. The three infective inocula produced symptoms of similar severity in maize. Bioassay of systemically infected leaves indicated the virus was equally infectious regardless of inoculum. In Southern blots of bioassay plants, no differences in MSV genome restriction endonuclease sites were observed. Thus, inoculation with the free linear or circularized MSV unit-length genome produced infections similar to those with plasmids carrying tandemly repeated genomes. The infectivity of free linear MSV unit-length genomes will facilitate molecular analysis of MSV, because cloning steps are minimized.