Aseptic udder quarter secretion samples (n = 1112) from 200 mastitic and 65 non-mastitic control heifers from 160 different farms were collected for bacteriological examination and in vitro antimicrobial susceptibility testing. Coagulase-negative staphylococci (CNS) were the most frequently isolated bacteria (57·8%). The percentage of Staphylococcus aureus was 20·1, of streptococci 11·3 and of other pathogens 10·8. Staph. simulans, Staph. hyicus, Staph. xylosus and Staph. chromogenes were the most common CNS. Staph. simulans and Staph. hyicus were most frequently found in clinical samples after parturition, whereas other CNS were equally or more often found in control samples. Most CNS infections detected in the prepartum period were eliminated spontaneously or with antibiotic treatment during early lactation, but the infected quarters were more susceptible to new infection by other pathogens. Milk N-acetyl-β-glucosaminidase activities were measured to monitor changes in inflammation. Among the CNS infections, N-acetyl-β-glucosaminidase activities were highest for Staph. hyicus and lowest for Staph. xylosus in the lactating mammary gland of heifers. CNS were resistant to trimethoprim–sulphonamide, ampicillin and erythromycin. Staph. aureus was more resistant than CNS to β-lactamase-sensitive penicillins.