Specimens from the cervix or posterior vaginal fornix of 50 women attending a venereal disease clinic and from the urethra of 50 men with nongonococcal urethritis were cultured for Mycoplasma hominis. Recovery of the organism in two conventional mycoplasma culture media containing horse serum was compared with that in SP-4 broth medium (which is used for the culture of fastidious mycoplasmas). M. hominis was isolated from approximately 63% of the specimens from women by means of either of the horse serum media and from 94% by means of SP-4 medium. The organism was isolated from 10-12% of the urethral samples from men by means of either of the horse serum media and from 22% by means of SP-4 medium. In all, 55 specimens yielded M. hominis in SP-4 medium and 37 in horse serum broth. Thus, 18 (33%) of the 55 isolates were recovered only when SP-4 medium was used. The presence of thallium acetate in the horse serum medium used in one laboratory appeared to contribute to its inferiority. These results show that SP-4 medium may be superior for the isolation of M. hominis in some circumstances. However, major modifications in this medium will be required for the selective recovery of Mycoplasma genitalium from the human urogenital tract when M. hominis is also present.