The aim of the study was to determine the association between PRL responses to suckling and maintenance of postpartum amenorrhea among breastfeeding mothers. Three blood spot samples (5, 30, and 50 min following a timed nursing bout) were collected from 71 intensively breastfeeding Nepali women for PRL determination. Maternal age, BMI (weight/height2), menstrual status, caste, infant age, nursing bout length, and duration of supplementation were recorded at time of sample collection. Independent and paired t tests, linear regression analyses, and general linear models were used to evaluate differences between cycling (n = 36) and amenorrheic (n = 35) women and associations among variables. Logistic regression analyses were used to relate PRL measures to the odds of maintaining lactational amenorrhea. Amenorrheic breastfeeding mothers had higher (P < .001) PRL levels at all 3 collection times than cycling breastfeeding mothers, and PRL levels declined with time since birth (P < 0.05). The odds (OR) of having ceased lactational amenorrhea was significantly higher (OR = 5.0, 95% Cl = 1.3-19.9) among mothers with lower PRL levels (< or = 10 ng/mL) at 50 min post-sucking, and PRL at 50 min showed a significant dose response relationship with menstrual status. The association between 50 min PRL levels and lactational amenorrhea appears to be independent of time postpartum, maternal age, BMI, nursing bout length, and duration of supplementation. Among intensively nursing women, maintenance of elevated PRL levels across the interbout interval increases the odds of maintaining lactational amenorrhea.