Bacterial L forms were isolated from milk samples of dairy cattle infected experimentally with Staphylococcus aureus. Initially, bacterial L forms were induced in vitro from 12 of 44 S. aureus strains isolated from bovine mastitis. Cows were experimentally infected in two experiments with strains shown in vitro to be easily inducible to L form and with S. aureus Newbould 305. Each quarter of the mammary gland was infected with either 10(3) or 10(6) CFU of the test strains. Treatment was initiated with 100,000 U of penicillin G per quarter at the first signs of clinical mastitis. Milk samples were collected daily and cultured on bovine blood agar and PPLO agar (Difco Laboratories, Detroit, Mich.) with 10% horse serum and 5% NaCl. Staphylococcal L forms were isolated from milk samples collected from infected glands in both experiments after antibiotic therapy. Glands with the highest concentrations of leukocytes and bacteria were most likely to yield L forms in milk samples after treatment was initiated. Cows harboring L forms typically yielded parental organisms after cessation of antibiotic therapy. No detectable changes occurred in antibiotic susceptibilities, coagulase production, or biochemical activities in strains induced to L form followed by reversion to the parental form. These results demonstrated that L forms can occur during treatment of bovine mastitis and that L forms may be one explanation for the poor response of staphylococcal bovine mastitis to antibiotic therapy.