Background: In vitro and in vivo analyses differ between the number of milk ducts found in the lactating breast, and there is a lack of knowledge as to whether or not external factors in the mother or the child affect the number of ductal orifices. The aim of this study was to determine the number of milk duct orifices in vivo and to investigate the possible influence of variable parameters in mother and infant. Methods: Study design: Prospective clinical trial. In 98 breastfeeding women we investigated the nipple surface in order to identify the number of milk duct orifices using Marmet’s manual milk expression technique. In addition mothers were interviewed on different parameters of birth and breastfeeding. Results: Every nipple had 3.90 ± 1.48 milk duct orifices on average. There was no significant difference between left and right breasts. The use of a breast pump in addition to breastfeeding did not have any effect on the number of ductal orifices. Multiparous women exhibited more ductal orifices (8.5 ± 3.0) as compared to primipara (7.1 ± 2.7). Boys were associated with significantly more ductal orifices in their mother’s right breast (4.2 ± 1.7) than girls (3.5 ± 1.4). Furthermore boys were breastfed for longer per session. A shorter birth height of males correlated with more ductal orifices in left nipples. Fluid intake of mothers was associated with a higher number of ductal orifices. Restless infant behavior could not be explained by less milk duct orifices. Pain in the breast during breastfeeding did not have an influence on ductal orifices either. Psychological criteria, such as duration of maternity leave and total intended breastfeeding period, did not affect the number of orifices in the papilla mammaria of breasts during lactation. Conclusion: For the first time an in vivo investigation of the number of ductal orifices in lactating women was conducted non-invasively and associations with variables in the mother and the child, birth parameters in infants, and breastfeeding parameters in mothers and children were assessed. We conclude that the number of activated ductal orifices on the surface of the nipple is primarily associated with functional aspects.