In order to determine the spectrum of CF mutations in the Spanish population, we have analysed 40 unrelated Spanish CF patients, with at least one chromosome negative for mutations delta F508, G542X, and N1303K. Exons 1-7,10-14a,15,16,17b,18-21 of the CFTR gene were studied by Single Strand Conformation Polymorphism (SSCP) analysis, using 60 known CF mutations as controls. SSCP screening allowed us to detect 28 different mutations in 52 CF chromosomes, and to identify four new mutations (Q30X in exon 2, A120T in exon 4, 1812-1G-->A in intron 11 and and 3667del4 in exon 19). Further analysis of the four new mutations in a total of 950 Spanish CF chromosomes showed a final frequency of 0.4%, 0.1%, 0.1%, and 0.1% for 1812-1G-->A,Q30X, A120T, and 3667del4, respectively. No mutations were detected in exons 1, 3, 14a, 16, and 18. We have also detected 10 intragenic polymorphisms and DNA sequence variants and have analysed their frequencies in our population. The total of 28 mutations identified in the 80 CF chromosomes highlight the molecular heterogeneity of CF in the Spanish population.