Two hundred nine men were studied to determine the optimal method of obtaining cultures for genital mycoplasma. Ureaplasma urealyticum (T-mycoplasmas) was isolated from 95 (45.5%) of the participants. Urethral cultures obtained by means of urethrogenital calcium alginate swabs identified 82 (86%) of the 95 colonized men. Urethral cultures taken with cotton-tipped applicators (76%) urine cultures (27%), and cultures of the coronal sulcus (24%) detected fewer colonized men. All men who were colonized with U. urealyticum were identified by one of the two urethral cultures. Mycoplasma hominis was recovered from 73 (34.9%) of the 209 men. Urethral cultures identified most of the circumcised men who were colonized with M. hominis (11 of 14; 79%). In contrast, cultures from the coronal sulcus detected most of the colonized uncircumcised men (49 of 59; 83%). More than 90% of the men who were colonized with M. hominis were identified by either urethral culture or culture of the coronal sulcus. A similar study was conducted among 143 normal college students yielded comparable results.