Recent evidence suggests that the susceptibility to respiratory distress syndrome (RDS) is partly explained by genetic variation in the surfactant proteins (SP) SP-A and SP-B. The present study was designed to evaluate the concordance difference method and candidate gene analysis, in parallel, for the investigation of genetic susceptibility to RDS. We studied 100 same-sex twin pairs with established RDS in at least one twin. The difference in RDS concordance rates between the monozygotic (MZ) and dizygotic (DZ) twin pairs as evidence of a genetic influence was evaluated, and the SP-A and SP-B genes were investigated for potential associations with the susceptibility to RDS. The concordance rates of RDS were 54 and 44% in the MZ and DZ pairs, respectively. The concordance difference of 10% was not significant [95% confidence interval (CI) -0.1 to +0.3, P=0.32], suggesting a low hereditary impact. However, the SP-B Ile131Thr polymorphism was associated with RDS. The threonine allele was associated with an increased risk of RDS [odds ratio (OR) 2.2, 95% CI 1.4-3.5, P=0.0014]. This was particularly apparent in first-born male infants (OR 6.2, 95% CI 2.4-16.3, P<0.001). The degree of prematurity (<32 weeks OR 2.0, 95% CI 1.1-3.7, P=0.021) and birth order (second-born OR 3.1, 95% CI 1.3-7.4, P=0.009) were the clinical variables affecting the risk of RDS. An association between the SP-B Ile131Thr polymorphism and RDS was found. The threonine allele was associated with the risk of RDS, particularly in the first-born twin infants. The concordance difference between MZ and DZ twin pairs underestimates the genetic impact on the risk of RDS. The traditional twin concordance study is insufficient to evaluate genetic predisposition to RDS or other diseases that are confounded by the birth order or multiple pregnancy in itself.