Technical Abstract: Current methods to estimate milk production in beef cows can be time- consuming, labor-intensive and subject to high variability. The objective of the current experiment was to validate a deuterium oxide (D2O) dilution method of estimating milk production in cattle. In a preliminary study, blood was collected from five Holstein calves for baseline D2O measurements followed by an injection of 300 mg D2O/kg BW. Another blood sample was collected after D2O was allowed to equilibrate with body water for 2 h, and on each of the next five consecutive days, prior to feeding. Actual milk intake was measured by disappearance and D2O in plasma was measured by mass spectrometry. Milk intake was computed from the disappearance curve of D2O in blood plasma for each calf. Accumulated milk intake estimated by D2O dilution was highly correlated with actual milk intake. Objectives of the main experiment were to determine if 1) D2O dilution was comparable to a standard measure of milk production in beef heifers and 2) growth hormone (GH) response to GH-releasing hormone (GHRH) in heifers at weaning is predictive of subsequent milk production. Results indicate that the D2O dilution method is correlated to the weigh- suckle-weigh estimation of milk production and that GH response to GHRH in weanling heifers is positively related to their subsequent milk production. Deuterium oxide dilution in calves offers a novel approach to estimate milk production of the dam in typical beef cattle production settings.