Objectives: To determine whether the incidence and severity of retinopathy of prematurity (ROP) differs between infants conceived naturally and those born after assisted conception, and to analyze the impact of singleton versus multiple gestation on ROP. Design: Comparative case series. Participants: The study group consisted of 363 infants with a birth weight (BW) of ≤1500 g who were hospitalized in the neonatal unit of a single tertiary-care center between 1998 and 2000. Methods: Data on gestational age (GA), BW, type of pregnancy (singleton/multiple), and type of conception (natural/assisted) were recorded, in addition to the ophthalmological results. Ophthalmological examinations were performed routinely at 4 weeks and repeated later, depending on the severity of the findings. Main Outcome Measures: Presence and stage of ROP were compared between infants conceived naturally and those conceived by assisted technology, and between singleton and multiple-birth infants. Results: Mean GA at birth was 29.4 weeks (standard deviation, 2.5; range, 23-36). Two hundred four neonates (56.2%) were conceived naturally and 159 were conceived by assisted conception, either in vitro fertilization (IVF) alone (n = 119 [32.85%]) or IVF combined with drug treatment (n = 40 [11%]). Sixty-four infants in the natural conception group were the product of multiple pregnancies, as were 103 infants in the assisted conception group. Retinopathy of prematurity was noted in 159 of 363 infants (43.8%): 89 conceived naturally (71% singletons, 28% twins, and 17% triplets) and 70 born by assisted conception (70% singletons, 47% twins, and 33% triplets). There was no significant difference in either occurrence or severity of ROP between the natural conception and assisted conception groups. Singletons had a significantly higher rate of advanced ROP (stages II-III) (30.2%) than twins (23.1%), triplets, and quadruplets (10.6%) (P = 0.024). On multiple regression analysis, low GA and BW were the variables most significantly associated with ROP. Conclusions: In our sample, assisted conception per se did not appear to be a risk factor for ROP. Singleton babies with a birth weight of ≤1500 g were more prone to develop ROP stages II and III than twins or triplets. Gestational age and BW were the most significant factors associated with ROP.