Unintegrated circular proviral DNA of a type D retrovirus (PMFV) isolated from a permanent human cell line was molecularly cloned in the bacteriophage vector L47.1 and subcloned in the plasmid vector pGEM-2. A restriction endonuclease map of PMFV DNA was established using 10 different enzymes for single and multiple digestions of closed circular and cloned DNA molecules. By restriction endonuclease analysis cloned PMFV DNA represented full-length viral DNA with one long terminal repeat (LTR). The comparison of the physical map of cloned PMFV to those of other cloned type D retroviruses revealed closest homology to the map of retrovirus D/New England (pD398) and SAIDS retrovirus type 1 (SRV-1). The relatedness of PMFV to further type D retroviruses (Mason-Pfizer monkey virus, MPMV; SAIDS retrovirus type 2, SRV-2) was also demonstrated by cross-hybridization of cloned DNAs under different stringencies (i) using full-length genomic probes of PMFV, MPMV, and SRV-2 and (ii) by DNA sequence analysis of regions of the group specific antigen (gag) protease (prt), polymerase (pol), and envelope (env) genes.