In the present study we investigate the optimal methodology for determination of the nasal carriage rate of Staphylococcus aureus. Tests were performed on 91 healthy laboratory staff. The reproducibility of different sampling, transportation, storage and culture methods was examined. We compared sterile dry cotton wool swabs with sterile dry cotton wool swabs impregnated with charcoal and 5% blood agar plates with mannitol salt agar plates after different incubation periods. Finally, we investigated the detection rate for S. aureus following direct plating compared to storage in Stuart's transport medium for 7 days. There were no differences in isolation rates from the right or left nostril using either cotton or charcoal swabs. Charcoal swabs gave an increased isolation rate as compared to cotton swabs, and incubation in broth enrichment medium containing 6.5% NaCl also increased the isolation rate. Storage in Stuart's transport medium for 7 days gave an increase in isolation rate as compared to direct plating on blood agar. With mannitol salt agar plates the increase in isolation rate when incubation was performed for from 2 to 4, 2 to 7, and 4 to 7 days was 5.9%, 16.7%, and 11.5%, respectively. For the detection of S. aureus nasal carriers we find the use of charcoal swabs and Stuart's transport medium combined with cultivation on mannitol salt agar for 7 days to be the optimal method.