We have modified deproteinization methods and a number of spectrophotometric and bioluminescent methods in order to measure the concentrations of cellular metabolites in small volumes (< 0·3 ml) of sows' colostrum and milk. For the majority of the assays, recoveries ranged from 92 to 105%. The binding of ATP and UTP to a calcium phosphate–citrate–caseinate complex in milk, and the decrease in ATP (92%/h) and UTP (18·1 %/h) concentrations during in vitro incubation of the whey fraction suggested that it was unlikely ATP and UTP (both < 1 µM) could exist free in sows' milk. The mean concentrations (range) of cellular metabolites in milk (6–11 d post partum) were: glucose, 669 µM (220–1367); glucose 6-phosphate, 63·0 µM (27·6–101·4); glucose 1-phosphate, 18·3 µM (13·1–24·8); UDPglucose, 296 µM (170–494); UDPgalactose, 635 µM (230–945); lactose, 162 mM (124–187); UDP, 105 µM (85–130); UMP, 1760 µM (1326–2587); inorganic phosphate, 135 mM (1·4–29·3); ATP, <0·5 µM; ADP, 53·6 µM (10·5–171·25); AMP, 215 µM (61·6–491·6); cAMP, 22·3 µM (3·5–61·6); galactose, 198 µM (118–474) and fructose, 226 µM (172–283). Differences in the concentrations of glucose, glucose 6-phosphate, glucose 1-phosphate, UDPgalactose and cAMP between fore and hind milk samples indicated postsecretory changes in the concentrations of certain metabolites. Changes in the concentrations of metabolites during in vitro incubation of milk and of colostrum suggested that these postsecretory changes were probably due to the actions of enzymes present in mammary secretion. Collection of milk that has been most recently secreted (hind milk) will provide the most accurate indication of the intracellular concentrations of these metabolites. The concentrations of cellular metabolites in the milks from different species are compared.