To verify to what extent mutation analysis on blood spot could improve cystic fibrosis neonatal screening in an area with high allelic heterogeneity, we designed a special protocol. Spot trypsin estimation at birth, trypsin re-testing after 1 month, meconium lactase testing and mutation analysis of delta F508, R1162X and N1303K, were retrospectively clustered according to different patterns (trypsin/lactase/mutation; trypsin/lactase/re-testing; trypsin/mutation) and compared. The programme, which lasted 2 years (1993-94) and covered most of North-eastern Italy, included 95,553 screened newborns. Thirty-four affected babies were detected by screening and one by meconium ileus (incidence 1/2730). The combined use of trypsin, lactase and mutation analysis in cystic fibrosis neonatal screening permits a better sensitivity compared to the two other combinations (34 diagnoses vs 32 in both cases). Moreover, the higher specificity of the former method (false positives 42 vs 148) allows a reduction of recalls, which cause considerable anxiety. We confirm in trypsin-positive newborns an increased frequency of cystic fibrosis heterozygotes (1/17).