A simple microtitre plate assay is used to detect antimicrobial activity in clinical urine specimens and its potential as a screening tool is assessed. The assay is based on a colorimetric substrate, p-nitrophenyl-beta-D-glucopyranoside, in combination with a Bacillus subtilis strain to detect antimicrobial residues. The assay identified antimicrobial activity in 31% of the 527 clinical urine samples tested. The majority of the samples (65%) came from the community, with the rest comprising hospital inpatients (19%) and out-patients (16%). The results demonstrated that there is an association between gender and the presence of inhibitory substances, as 40% of males and 27% of females tested positive. Just over two-fifths of hospital patients (46%) tested positive for inhibitory substances, compared to 26% of samples from community patients. Of the 306 samples that were culture-negative (<10(4) bacteria/mL), 42% were positive for inhibitory substances, compared with 17% among the remaining 221 samples. However, there was no evidence of an association between age and the presence of inhibitory substances. This study demonstrates that the bacteriostatic effect of the bacterial preservative boric acid is sufficient to upset the specificity of the assay. Furthermore, it has been suggested that antimicrobial activity can confuse the interpretation of culture results, as they have been found to play a major role in the occurrence of apparently sterile pyuria.