Many laboratory workers prefer the rhodamine-auramine method of staining acid-fast bacilli (AFB) in primary specimen smears rather than carbol fuchsin stains because the stain is more readily interpreted and yields greater sensitivity. The increasing incidence of AFB infections serves as an impetus to optimize the rhodamine-auramine stain. A total of 782 primary smears were evaluated blindly by the rhodamine-auramine method at both room temperature and 37 degrees C. Thirty-five smears (4.5%) were positive for AFB, 30 were positive by both methods, and 5 were positive at 37 degrees C only. Room temperature staining detected only 85.7% of the positive primary smears. Of the 30 smears positive by both methods, 13 (43.3%) had equal numbers of AFB on both smears, 13 (43.3%) had more AFB on the smear stained at 37 degrees C, and 4 (13.3%) had greater numbers of AFB on the smear stained at room temperature. No smears were positive only when stained at room temperature. The increasing diagnostic emphasis placed on the primary smear underscores the importance of optimizing AFB smear methods, and rhodamine-auramine staining at 37 degrees C enhances the detection of AFB compared with conventional staining at room temperature.