Interactive genetic and environmental factors may influence the differentiation of surfactant and the risk of respiratory distress syndrome (RDS). DNA samples from 441 premature singleton infants and 480 twin or multiple infants were genotyped for surfactant-specific protein (SP)-A1, SP-A2, and SP-B exon 4 polymorphisms and intron 4 size variants in a homogeneous white population. Distributions of the SP-A and SP-B gene variants between RDS and no-RDS infants were determined alone and in combination. SP-A1 allele 6A2 (p = 0.009) and the homozygous genotype 6A2/6A2 (p = 0.003) were overrepresented in RDS of singletons when the SP-B exon 4 genotype was Thr/Thr, and underrepresented in RDS of multiples when the SP-B genotype was Ile/Thr (p = 0.012 for 6A2 and p = 0.03 for 6A2/6A2) or Thr/Thr (p = 0.12 for 6A2 and p = 0.018 for 6A2/6A2, respectively). The SP-A 6A2 allele in the SP-B Thr131 background predisposed the smallest singleton infants to RDS, whereas near-term multiples were protected from RDS. There was a continuous association between fetal mass and risk of RDS, defined by the SP-A and SP-B variants. Labeled lung explants with the Thr/Thr genotype showed proSP-B amino-terminal glycosylation, which was absent in Ile/Ile samples. Genetic and environmental variation may influence intracellular processing of surfactant complex and the susceptibility to RDS.