The concentrations of lactose, glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDPglucose (UDPglc), UDPgalactose (UDPgal), and inorganic phosphate (P(i)) (metabolites in the lactose synthesis pathway) were measured in mammary secretion from nondiabetic (ND) and insulin-dependent diabetic (IDD) mothers during the first 10 days postpartum to determine their relationship with the amount of lactose synthesized and their association with the delay in lactose synthesis in IDD mothers. For all mothers the concentrations of all metabolites were low initially, and in ND mothers the first increases occurred as follows: lactose--day 2; glucose, G6P and P(i)--day 3; G1P and UDPglc--day 4; and UDPgal--day 6. The first increases for IDD mothers occurred 1-4 days later than for ND mothers. The concentrations of glucose, G6P, G1P, UDPglc, and P(i) were related to the amount of lactose synthesized. Since the rate of lactose synthesis and concentrations of other metabolites were less than half-maximal while the concentration of glucose was low, and since there was a delay in the increase in mammary gland concentrations of glucose in IDD mothers, it is concluded that glucose availability has the potential to play a role in the regulation of the rate of lactose synthesis at lactogenesis II.