Mycoplasma virus P1 is a tailed, polyhedral virus isolated from Mycoplasma pulmonis. To characterize the P1 genome, stocks of virus were prepared free of host cell nucleic acids. A single DNA species of 11.3 kb that was shown by plaque hybridization to be of P1 origin was extracted from the virus. Efficient isolation of P1 DNA required digestion with proteolytic enzymes prior to phenol extraction. Although P1 DNA was double-stranded and linear following such treatment, it was resistant to digestion with the 5'-specific λ exonuclease. Electron microscopic analysis indicated that globular material is complexed to the ends of P1 DNA. The globular material was not observed on protease-treated P1 DNA molecules, suggesting that it is composed of protein. Removal of the putative terminal protein by chemical treatment allowed the cloning of the P1 DNA ends, and nucleotide sequence analysis revealed that these ends contain a 350-bp inverted terminal repeat. © 1995 Academic Press, Inc.