Acholeplasmas have been isolated from a variety of animals, insects, and plants, but only Acholeplasma laidlawii has previously been found in humans. We have isolated Acholeplasma oculi in pure culture from the amniotic fluid of a woman at 19 weeks of gestation. The organism was positively identified by growth inhibition, epi-immunofluorescence, and arbutin hydrolysis. Demonstration of organisms directly in amniotic fluid by DNA fluorochrome and immunofluorescence staining provided additional evidence that the isolate was genuine and not a medium contaminant. The remainder of the pregnancy was unremarkable, and a full-term male infant was delivered without complications. Even though there is some evidence possibly associating A. oculi with various diseases in livestock, the prevalence and significance of A. oculi in humans has not been determined. © 1987 Springer-Verlag New York Inc.