Screening of milk samples from healthy and mastitic cows showed that 3 of 136 samples from mastitic cows were positive for mycoplasma F-38; all the milk samples from the 145 healthy cows were negative for mycoplasma F-38. One of the mycoplasma F-38 isolates (CM-193) was recovered in pure colonial form, one in a mixed growth with Mycoplasma bovigenitalium and M. bovirhinis and one with M. bovigenitalium, M. bovirhinis and Acholeplasma laidlawii. The mixed growths also contained colonies of Staphylococcus epidermidis. The isolates were all sensitive in vitro to erythromycin, lincomycin, ledermycin, kanamycin, spiramycin, vibramycin, tetracycline and tylosin but were resistant to ampicillin and streptomycin. The pure isolate of mycoplasma F-38 (CM-193) was pathogenic to hamster tracheal epithelium and induced mastitis in rats.