The aim of this study was to elucidate the modulation of behaviour and endocrine stress responses of guinea-pig pups by social and spatial factors in a maternal separation paradigm. The animals were kept in two colonies (each colony: nine males, 13 females and their offspring; enclosure size: 6 m2). Blood samples were taken from the ear vessels of eight male and eight female pups (aged 13–14 d) immediately before and 2 h after they were removed from their colony and were placed singly into a novel environment. Furthermore, eight male and eight female pups were tested in their home colony before and after 2 h of sepration from their mothers. Blood samples were taken from control animals (eight male and eight female pups) which remained in the home colonies together with their mothers. Additionally, the behaviour of 16 male and 16 female pups was recorded in their home colonies when their mothers were either present or absent. Male and female pups separated from their mothers showed a significantly higher locomotor activity, with females showing higher frequencies of distress calls, than pups whose mothers were present. The mother's absence did not cause a significant increase in serum cortisol concentrations in the male or the female offspring. This result is entirely different from the endocrine stress response of pups that were placed singly in an unknown environment. These animals showed a significant increase in their serum cortisol concentrations after 2 h of separation from their mothers. The absence of the mother led to distinct changes in the behaviour of the guinea-pig pups. However, staying in the familiar social and spatial environment buffered the endocrine stress response that normally occurs because of maternal separation.