The objective was to characterize the antibody isotype responses to vaccination with a commercial Staphylococcus aureus bacterin in dairy heifers. Twenty-five Holstein-Friesian dairy heifers were assigned at random to one of two groups, vaccinates (n=14) or controls (n=11). Vaccinates received two 5-ml doses of Lysigin 28 d apart in late gestation. Both groups were challenged with a heterologous serotype 5 strain of Staph. aureus by aseptic intramammary infusion on days 6, 7 and 8 of lactation. Samples for serum antibody isotype (IgG, IgG1, IgG2 and IgM) determinations were taken before each vaccination, immediately prior to challenge (6 d post-calving) and at the end of the study (28 d post-calving). Samples for milk antibody isotype determinations were taken immediately prior to challenge and at the end of the study. Antibody isotype sample-to-positive ratios (S[ratio ]P ratio=(mean test sample OD−mean within-plate negative control OD)/(mean within-plate positive control OD−mean within-plate negative control OD)) were determined in milk and serum using a series of ELISAs coated with different strains of Staph. aureus belonging to capsular polysaccharide (CP) serotype 5 or 8 or surface polysaccharide (SP) serotype 336. Vaccinates had higher mean serum IgG1 and IgG2 S[ratio ]P ratios than controls against all three strains of Staph. aureus (P[les ]0·023). Vaccinates had higher mean milk IgG S[ratio ]P ratios than controls against CP8 and SP336 strains of Staph. aureus (P[les ]0·030). Hence, a humoural immune response to the vaccine was detected in serum and milk, but responses varied according to strain and antibody isotype tested.