The mechanisms that regulate mammary blood flow during lactation are not fully understood. In the present study laser Doppler flowmetry (LDF) was used to measure blood flow in the cutaneous microvessels of the mammary gland of lactating rats. The effects of suckling on blood flow were examined, as were those of local injection of oxytocin (0.5–5 mU) and the vasoactive peptides calcitonin gene-related peptide (CGRP; 0.1–10 pmol), vasoactive intestinal polypeptide (VIP; 0.4–20 pmol) and neuropeptide Y (NPY; 1–40 pmol). Blood flow responses to suckling varied depending on how much time had lapsed since the previous suckling. In rats with milk in the gland, suckling caused an initial increase in blood flow. In connection with milk let-down, the blood flow decreased, but was followed by a second increase. In recently suckled rats with no milk in the gland the increase in blood flow corresponded to the number of pups suckling. Oxytocin injections also had varying effects on mammary blood flow depending on how recently suckling had taken place. In non-suckled rats with milk in the gland, oxytocin injections caused a rise in blood flow that was interrupted by a fall during milk ejection. In recently suckled rats, all doses of oxytocin caused an increase in blood flow of similar magnitude. However, the effect of the higher doses had a longer duration. CGRP and VIP injections caused a dose-dependent increase in mammary blood flow regardless of when suckling last occurred. NPY injections caused a dose-dependent decrease in blood flow.