The aim of the present study was to develop a broad-range PCR based on bacterial 16S rDNA for use in the routine diagnostic clinical microbiology service. The optimization and validation of the assay for use on clinical specimens from normally sterile sites is described, and preliminary results are reported on the use of the assay in the clinical diagnosis of bacterial infection in 382 paediatric specimens over a 2-year period. These results are compared to those obtained by standard culture techniques and show increased diagnosis of bacterial infection when both culture and PCR are used together; 16S rDNA PCR provided the sole evidence of pathogenic infection in 71 cases. Key stages in the assay development and potential pitfalls of the technique are highlighted and the improvement the assay offers in the diagnosis of infection in the paediatric setting is discussed.