A better understanding of how biological processes affect milk production in dairy cows is important to improve lactation persistence. The present study investigated the sensitivity of the mammary gland to prolactin. Eleven mid-lactation cows were milked twice daily during the first 2 experimental weeks. During wk 3–10 cows were differentially milked; the right quarters being milked thrice daily (3×), while the left were milked once daily (1×). During wk 11–14, all quarters were milked twice daily. After 4 weeks of differential milking, cows received daily i.m. injections of the dopamine antagonist domperidone (300 mg; DOMP; n = 6) or DMSO (CTL: n = 5) for 8 weeks (wk 7–14). Blood and milk samples were collected weekly for measurement of prolactin (PRL) as well as milk components for the latter. Milk was also collected from front quarters every 2 weeks and RNA was extracted from milk fat fraction. Expression of PRL receptors (long; PRLRL and short; PRLRS) were measured by real-time RT-PCR. Mammary gland biopsies were taken from rear quarters on wk 2, 6, 10 and 14 for subsequent gene and protein analyses. Differential milking improved milk production in the 3× quarters compared with the 1× quarters (P < 0.01). Increases in fat (P = 0.04), protein (P = 0.05) urea (P = 0.01) and lactose (P < 0.01) were observed in the 1× quarters, while greater SSC (P < 0.01) was observed in the 3× quarters. Blood PRL concentrations were greater for DOMP than CTL cows (24.8 ± 2.3 vs 13.9 ± 2.5 ng/mL; P < 0.01). There was no effect of injections on milk production, but milk from DOMP cows had greater concentrations of lactose (P = 0.02) than CTL. Interestingly, DOMP cows had greater expression of both forms of PRLR (P < 0.01) in milk fat compared with CTL. Additionally, greater PRLR expression was observed in the 3× than 1× quarters (P < 0.01). Results show that milk frequency improves milk production and alters composition and expression of PRLR in the mammary gland. Moreover, administration of domperiodone increased PRL concentration and is related to greater expression of both forms of PRLR in the mammary gland.