A miniaturized sensitive bioassay was used to detect tetracycline in open comedones following topical twice daily application of 0.22% tetracycline hydrochloride for a minimum of 4 weeks to the facial skin of patients with mild to moderate acne. The lower limit of detection was 4.8 +/- 0.8 ng per comedone or per 10 microliters. Using this method, 111 of 155 open comedones from 15 patients were found to contain a detectable amount of tetracycline, ranging from 1.8 to 156.9 ng per comedone, and between 4.5 and 1140.1 ng per mg comedonal material. There was a significant effect of comedone weight on tetracycline content, with smaller comedones containing proportionately more tetracycline. The Spearman rank correlation coefficient was -0.5619 (P < 0.001). All 111 comedones in which tetracycline was detected contained sufficient drug to inhibit fully antibiotic-sensitive propionibacteria. However, conditions favourable to the selection and overgrowth of highly tetracycline-resistant strains (MIC > or = 32 micrograms/ml) prevailed in at least 18.7% (29 of 155) of the comedones tested.