DNA isolated from Mycoplasmatales viruses MVL51 and MVGs51 was infectious when mixed with Acholeplasma laidlawii BN1-Na1 R cells. Infectivity was destroyed by deoxyribonuclease but not by ribonuclease, Pronase, or specific antiserum to the virus. Host mycoplasma cells were only competent for transfection during late-log growth phase. The rates of the establishment of DNase insensitivity of viral DNA transfectants were similar to those of bacteriophage systems. The dose-response curve for transfection suggested that an average of six molecules of DNA must interact with a cell in order to produce one infectious center. Mycoplasmatales virus DNA exhibited a low efficiency of infection; one infectious center required 4 × 10 5 virus equivalents of DNA.