The effect of different diaper care procedures on skin barrier function in infants has been minimally investigated and may be assessed using objective methods. In a single-center, prospective trial, 89 healthy 9-month-old infants (±8 wks) were randomly assigned to three diaper care regimens: group I used water-moistened washcloths at diaper changes (n = 30), group II additionally applied diaper cream twice daily (n = 28), and group III used wet wipes and diaper cream twice daily (n = 31). Transepidermal water loss (TEWL), skin hydration (SCH), skin pH, interleukin 1α (IL-1α) levels, and microbiologic colonization were measured in diapered skin (upper outer quadrant of the buttocks), nondiapered skin (upper leg), and if diaper dermatitis (DD) occurred, using the most affected skin area at day 1 and weeks 4 and 8. Skin condition was assessed utilizing a neonatal skin condition score and diaper rash grade. On diapered skin, SCH decreased in groups II and III, whereas TEWL values were reduced in group II only. Skin pH increased in groups II and III. In general, SCH, skin pH, and IL-1α levels were higher in healthy diapered skin than in nondiapered skin. The incidence and course of DD was comparable in all groups. Areas with DD had greater TEWL and skin pH than unaffected skin areas. Infants who received diaper cream had lower SCH and TEWL and higher pH levels in the diapered area than on nondiapered skin. No correlation with the occurrence of DD was found.