The objective was to investigate the changes occurring in the activities of the enzymes lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in sheep and goat milk as a result of subclinical intramammary infections (IMI) and to evaluate the use of these enzymes for the diagnosis of subclinical IMI in dairy sheep and goats. A total of 206 samples of sheep milk and 162 samples of goat milk, obtained from equal udder halves, were used in the study. For each species they were divided into two groups: a no-infection group and a subclinical infection group. Activities of LDH, ALP and AST were significantly higher in the subclinical infection group than in the no-infection group (P<0·05) in both sheep (LDH: 350·42±11·25 v. 120·91±4·41; ALP: 2773·43±105·18 v. 2189±94·24; AST: 29·57±0·74 v. 17·32±0·46) and goats (LDH: 354·07±13·33 v. 103·79±3·75; ALP: 311·13±25·74 v. 137·24±19·62; AST: 27·59±6·42 v. 15·87±0·45). The activity of LDH was identified as indicator for subclinical IMI in both sheep and goats. The optimum cut-off values for LDH activity, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic (ROC) analysis, were at 197 U/l, 185 U/l and 197 U/l for sheep, goats and both species, respectively. DSn for sheep, goats and both species at these cut-off values was 92·8%, 98·2% and 94·0%, whereas DSp was 95·4%, 96·3% and 96·3%, respectively. It was concluded that the determination of LDH activity in milk serum is a sensitive and reliable method for the detection of subclinical IMI in dairy sheep and goats.