The ability of molecular clones of human T-cell leukemia virus type I (HTLV-I) to direct the synthesis of infectious virions has not previously been demonstrated. An HTLV-I provirus originating from an adult T-cell leukemia patient was cloned into a plasmid vector and is designated pCS-HTLV. This molecular clone was shown to direct the synthesis of viral mRNA and proteins in transiently transfected cells; in addition, virus structural proteins were released into the culture medium. Viral proteins were assembled into virions that sedimented at a buoyant density characteristic of retrovirus particles and whose morphology was verified by electron microscopy. Virions concentrated from transiently transfected cell supernatants were incubated with primary cord blood lymphocytes or with transformed T-cell lines to establish that these particles were infectious. Expression of spliced, viral mRNAs in the T-cell cultures after both primary and secondary infections with cell-free virus revealed that pCS-HTLV encodes an infectious provirus.