We profiled total mammary blood flow and trans-mammary disappearance of metabolites in heat-stressed lactating dairy cows. Eighteen mid-lactation Holstein cows were housed in tie-stalls in environmental chambers and subjected to one of the 3 treatments: (1) hyperthermia and ad libitum feed intake (HS), (2) normothermia and pair-feeding relative to HS (PF), or (3) normothermia and ad libitum feed intake (CT). All cows were housed under CT for 5 d before onset of treatments. Hyperthermia was modulated over 14 d of constant heat stress by increasing THI to ~80, thereby elevating rectal temperatures (RT) to ~40C, then maintaining RT at that level. Cows on normothermia were exposed to constant thermoneutrality (THI~66). After hyperthermia, HS cows were returned to CT conditions for 8 d to evaluate recovery. Total mammary blood flow was measured throughout the trial by transrectal scanning of both right and left external pudic arteries using color Doppler ultrasound equipped with a convex probe. Mammary arteriovenous blood samples were collected, analyzed for concentration of metabolites, and trans-mammary metabolite disappearance was calculated. During hyperthermia, mean RT was higher (P < 0.001) in HS than PF and CT (40.1, 38.4 and 38.5 ± 0.1°C, respectively), DMI was reduced similarly (P < 0.001) in HS and PF, relative to CT (11.0, 11.2 and 19.8 ± 0.8 kg/d, respectively), and milk yield was reduced more (P < 0.10) in HS than in PF, relative to CT (18.2, 21.1 and 27.8 ± 1.4 kg/d, respectively). Similar to DMI, total mammary blood flow decreased (P < 0.002) in HS and PF compared with CT (405, 363 and 557 ± 36 L/h, respectively). Relative trans-mammary disappearance of glucose was similar between PF and CT but higher (P < 0.01) than HS (23.0, 22.4, 16.5 ± 1.8%, respectively). Relative trans-mammary disappearance of triglycerides did not differ between treatments, averaging 46.5 ± 2.6%. For all variables measured, HS and PF cows returned to CT levels within 8 d of recovery. Heat stress reduces total mammary blood flow and trans-mammary disappearance of metabolites in lactating dairy cows.