Preterm and term transitional milks of human subjects and mature milks of human subjects, non-human primates and non-primates were analysed for free amino acids (AA) using precolumn phenylisothiocyanate derivatization and liquid chromatography. Differences in free AA between three types of human milk were small. Milks of pinnipeds (seals and sea lions) contained the highest levels of total free AA (8634–20 862 μmol/1), while the milks of cows and sheep had the lowest levels of total free AA (1061–1357 μmol/1). The milks of human subjects, chimpanzees (Pan troglodytes), gorillas (Gorilla gorilla), elephants (Elephas maximus), horses and pigs had intermediate levels of total free AA (3069–7381 μmol/1). Glutamic acid was the most abundant free AA in milks of human subjects (1339–2157 μmol/1), non-human primates (423–2528 μmol/1), elephants (1332 μmol/1), horses (1119 μmol/1), and cows (349 μmol/1). Taurine was the most abundant free AA in milks of pinnipeds (5776–13 643 μmol/1), pigs (1238 μmol/1), goats (1150 μmol/1) and sheep (341 μmol/1). Taurine was the second most abundant free AA in milks of human subjects and non-human primates, while histidine was the second most abundant free AA in milks of pinnipeds. Milks of each species had a distinctive free AA pattern which may reflect the relative importance of the free AA during early postnatal development.