SETTING: Ten peripheral laboratories performing routine acid-fast bacilli (AFB) smear microscopy in Lima, Peru. OBJECTIVES: To test whether external quality assessment (EQA) rechecking of AFB smears becomes more efficient with stratified lot sampling of treatment followup smears. DESIGN: In 2 consecutive years, a stratified lot sample of 36 treatment follow-up slides and 24 diagnostic slides were randomly selected and blindly rechecked. A second controller determined the final result for discordant slides. Feedback was provided to laboratory technicians during supervisory visits. RESULTS: More false-negative errors were found in the follow-up slides than in the tuberculosis suspect slides: 25 vs. 3. This represented a yield of 3.5% in 720 followup slides and only 0.6% in 480 diagnostic slides. Positive predictive values were high in both years. Respectively three and eight laboratories did not reach a relative sensitivity of >65% during the first and second year, and a clear improvement was seen in only one laboratory. Excessive workload seemed to preclude raising the level of routine performance. CONCLUSIONS: EQA with stratified lot sampling of treatment follow-up slides proved very efficient and effective for identifying laboratories with substandard performance in a setting with low positivity rates in routine diagnostic smears.