We investigated whether the inhibition of milk ejection during and/or between machine milkings is responsible for the low milk fat observed in commercial milk obtained from dairy ewes managed with a mixed system (MIX) of partial daily suckling (10 h) and once daily machine milking (after 14 h of udder filling). East Friesian crossbred dairy ewes were randomly allocated postpartum to the MIX system (n = 9) or to exclusive twice-daily machine milking (DY1, n = 8). Following wk 4, MIX ewes were permanently weaned from their lambs and milked twice daily. All ewes were injected with saline, oxytocin, or an oxytocin-receptor antagonist prior to three morning milkings during wk 2, 4, and 6 of lactation to study cisternal and alveolar milk distribution. Overall milk yield (cisternal + alveolar) for MIX ewes was 42% greater than for DY1 ewes during wk 2 and 4, which demonstrates the beneficial effect of lamb suckling on milk production of dairy ewes. However, during normal machine milking, only the cisternal fraction was obtained from MIX ewes, confirming that milk ejection did not occur for as long as these ewes remained in partial daily contact with their lambs. Although the volume of milk stored within the cistern, and its concentration of milk protein was similar for the two weaning systems, milk of MIX ewes was significantly inferior in cisternal milk fat concentration and yield compared to DY1 ewes. This provides evidence that not only is there inhibition of milk ejection during machine milking of MIX ewes, there is additional inhibition of transfer of milk fat, but not milk protein, from the alveoli to the cistern during the evening when MIX ewes are separated from their lambs. Following weaning of MIX ewes, the majority of lactation traits studied were similar compared to DY1 ewes.