Parathyroid hormone-related protein (PTHrP) has been shown to be present in milk of various mammals. We have assayed PTHrP in milk of various species by radioimmunoassay and estimated the molecular weights by Western blot analysis. PTHrP concentrations in bovine, ovine and human milk were 59.2 +/- 18.5, 74.1 +/- 35.0 and 36.6 +/- 20.7 micrograms/l (mean +/- S.D.) respectively, in pooled samples collected at various stages of lactation. PTHrP in mammalian milk was found to exist in two forms with molecular weights of 17.5 kDa and 21.5 kDa approximating those of PTHrP(1-108) and (1-141) respectively. In comparison, marsupial milk PTHrP appeared as a single low molecular weight form of 14.4 kDa which approximated to that of PTHrP(1-84). We performed a longitudinal study measuring the concentration of PTHrP in milk throughout lactation in cows, and found it to increase with the duration of lactation (r = 0.669, n = 91). We further examined the relationship between the concentration of PTHrP and total calcium in bovine milk, and the differences between these constituents in milk from Friesian and Jersey cows. PTHrP concentrations correlated positively with total milk calcium (r = 0.346, n = 105). The mean milk concentration of PTHrP of the Jerseys was significantly higher than that of the Friesians (52.6 +/- 5.4 micrograms/l compared with 41.0 +/- 4.8 micrograms/l, P less than 0.01), as was the mean milk calcium concentration (30.5 +/- 3.0 mmol/l compared with 26.7 +/- 2.7 mmol/l, P less than 0.01). We therefore postulate that production of PTHrP by the mammary gland may be associated with calcium transport from blood to milk. Also PTHrP may play a role in the development of milk fever in Jerseys which are predisposed to this condition.