Staphylococcus aureus mastitis is an important cause of economic loss for the dairy industry. Control programs rely on the timely and accurate identification of positive quarters. The effects of sampling time and sample handling were examined in an attempt to improve the accuracy of detection of S. aureus. Premilking and postmilking milk samples were collected from 55 lactating quarters with subclinical S. aureus infection. Each sample was divided into 2 aliquots; one of which was cultured fresh, the other was frozen at -20 degrees C for 14 days before being cultured. Analysis of variance was used to determine the effect of sampling time (premilking vs postmilking) and sample handling (fresh vs frozen) on the detection of S. aureus, as measured by the mean category for colony-forming units per millilitre (cfu/mL). A stratified analysis was required, due to interaction between sampling time and sample handling. Only a fresh postmilking sample was inferior, yielding a lower mean category for cfu/mL (P < 0.05). The ability to detect S. aureus in quarters with subclinical intramammary infection, as measured by the mean category of cfu/mL, was maximized in fresh or frozen premilking samples and in frozen postmilking samples.