It is believed that milk production is determined by the number and activity of mammary secretory cells. Secretory activity, as assessed by milk volume, depends on secretion of the major osmole in milk, lactose, which is produced by lactose synthase. The amount of either of the two proteins in lactose synthase may regulate milk production. The objective of this study was to determine whether the concentrations in milk of the two components of lactose synthase, α-lactalbumin (α-LA) and β1,4-galactosyltransferase (B4GALT), were related to genetic background, stage of lactation, breed or parity of dairy cows. α-Lactalbumin and B4GALT concentrations were measured by ELISA and by enzyme assays, respectively, from single milk samples. Two herds with a total of 279 cows were used in the analysis. One herd contained Ayrshire, Brown Swiss, Holstein and Jersey cows; the second herd contained two groups of cows; Holsteins selected for high milk production and Holsteins with 1960s genetics. The α-LA concentration in milk was greater in Jerseys and Ayrshires than in Holsteins and Brown Swiss. However, no difference in α-LA concentration was observed in milk from high and low genetic merit cows in the Minnesota herd or among different genetic backgrounds in the Illinois herd. β1,4-Galactosyltransferase concentrations were similar for all groups that were analyzed. α-Lactalbumin concentrations were positively correlated with milk protein concentration, milk fat concentration and lactose concentration. β1,4-Galactosyltransferase concentration in milk exhibited a strong positive correlation with number of days in milk. Although the concentration of B4GALT increased as lactation progressed, the values did not show any correlation with persistency of lactation or late lactation milk production. In conclusion, this survey shows that the two components of lactose synthase are each correlated to protein concentration and individually correlated to the concentration of other milk components and stage of lactation.