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The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322. The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice. Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule.