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Previous attempts to show a quantitative relationship between maternal hormone levels and early milk output have used small sample sizes and simple correlations. Women of mixed parity and similar socio-economic status and education were recruited to a study using multivariate analysis to look for these associations. Hormone levels (oestradiol, progesterone, prolactin and thyrotropin (TSH)) were determined for 91 mothers at four time points (ante- and postnatally) from finger-prick blood spots by fluoro-immunoassay. Milk output at 1 and 4 weeks was determined from 24-h test weighings. Parity was found to be the most significant factor affecting breast milk volume at 1 wk postpartum (multiparous women delivered 142 ml more milk in 24 h than primiparous women). Total time spent feeding had a strong association with breast milk volume, with increasing time having a negative effect. Multiple regression analysis, controlling for parity and time spent feeding, showed a positive association of milk output at 1 wk with antenatal progesterone and antenatal prolactin levels. At 4 wk, higher postpartum oestradiol levels had a negative association and antenatal progesterone levels a positive association with milk output. This study demonstrates that there are quantitative associations between antenatal maternal hormone levels and breast milk output in the early postnatal period. □Breast milk output, oestradiol, parity, progesterone, prolactin