The objective of this study was to examine the effect of breed, parity and body fatness on the lipolytic response of dairy cows. The lipolytic response was estimated as the plasma non-esterified fatty acid (NEFA) response to an adrenergic challenge. Four challenges per lactation were carried out on 124 Danish Holsteins, 101 Danish Red and 82 Jerseys through consecutive lactations. Within breed, there were two genetic lines. Cows were equally distributed across two feeding treatments, a normal and a low energy total mixed ration. Diet composition was constant throughout lactation. The lipolytic response was significantly affected by breed, parity and stage of lactation. Lipolytic response was greater in early lactation than other stages in lactation (P 0·001). The larger breeds had a higher lipolytic response than Jerseys (P 0·05), and showed an increase in lipolytic response between first and second parity (P 0·05), but not between second and third lactation. Lipolytic response in Jerseys was not significantly affected by parity. No line or feeding treatment effects were observed on the lipolytic response. Ultrasound measurement of the area of subcutaneous backfat was used to estimate effect of body fatness on the lipolytic response. There was an increase in lipolytic response with increasing body fatness (P 0·05) in mid lactation and the dry period. The slope of this was not affected by breed or parity. It was concluded that the lipolytic response of dairy cows, and by implication the responsiveness of the lipid reserves, varies according to breed, parity and physiological state.