Nontuberculous (i.e., atypical) mycobacterial infections are increasing among pediatric and immunosuppressed patients, who commonly present with subcutaneous inflammatory masses or adenitis, which is often surgically excised. Since the most frequently isolated species also grow slowly in culture, early diagnosis may depend on histologic detection of a mycobacterial organism in the biopsy specimen. However, the histologic methods used for this purpose are of uncertain value in the diagnosis of these infections. Biopsy specimens from 22 patients with clinical histories highly consistent with nontuberculous mycobacteriosis in which part of the tissue was cultured were selected for study. Coded tissue blocks and control specimens were stained by the Ziehl-Neelsen (ZN) or auramine-O (A0) fluorescent technique and examined blindly for the presence of characteristic organisms. Results of these studies were compared with the culture results, and predictive values were calculated. This experience showed that the AO technique is technically simpler, allowing faster screening at lower power and showing greater sensitivity and predictive value of a negative result although less specificity than the ZN technique. The lower specificity of AO may be factitious and due to the detection in the tissue of organisms that did not grow in culture. Previous observations that nontuberculous mycobacterial infections may elicit tissue reactions that simulate cat-scratch disease, sarcoidosis, and nonspecific chronic inflammation were also confirmed.