Two Mycoplasma strains recovered from tissue culture environments were found to grow in complex media devoid of serum or serum fractions containing cholesterol and in a cholesterol-free synthetic medium. Neither strain was capable of synthesizing pigmented carotenoids, although these compounds are present in, and characteristic of, other sterol-nonrequiring mycoplasmas. Serological tests and an analysis of their cell protein patterns obtained by gel electrophoresis indicated that the isolates were similar to each other but distinct from other sterol-nonrequiring serotypes, Mycoplasma laidlawii and M. granularum, as well as from sterol-requiring species. The existence of Mycoplasma other than M. laidlawii and M. granularum without sterol requirements suggested the need for some taxonomic changes in this group of organisms.