Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasian populations. The Spanish CF population is highly heterogeneous, with more than 70 different mutations causing CF. Since the CFTR gene was cloned, we have performed 81 prenatal diagnoses for 74 couples. Sixty-nine cases had a high risk (1/4) for C F and 12 presented a lower risk (11240). Direct analysis was possible in 36 cases (44.4 per cent); it was necessary to combine mutation analysis with polymorphic markers in 24 cases (29.6 per cent); mutation analysis and microvillar enzymatic (MVE) analysis were combined in five cases (6.1 per cent); and in 16 cases (19.8 per cent), only indirect analysis was possible. Nine different mutations were detected in this series of families: 621 + l G -+ T , dF508, 1609delCA, G542X, G551D, 1949de184, R1162X, W1282X, and N1303K. Another ten mutations were identified in these samples after prenatal diagnosis (181 1 + 1.6kbA-G, 71 1 + I G -P T , 2869insG, G85E, 2176insC, dI507, 3272-26A+G, Q890X, R1066C, and 4005+ 1G-PA). Our current strategy for molecular diagnosis of CF in the Spanish population is based, as a first step, on direct analysis for the two most frequent mutations (dF508 and G542X) and indirect analysis using the intragenic markers IVS8CA, IVSl7BTA, and IVSI7BCA. The second step consists of screening for the mutations already associated with the CFTR microsatellite haplotypes. The third step is a specific search for unknown mutations. While actual diagnostic methods are not automatic and robust enough for heterogeneous populations, the diagnostic strategy outlined provides rapid, accurate, and reliable prenatal diagnosis for the majority of couples.