To screen for point mutations causing protein S deficiency, we used a sequence of techniques specifically for the study of the protein S active gene, PS alpha. This strategy comprises amplification of exons and intron/exon junctions by means of the polymerase chain reaction (PCR) and electrophoresis of the amplified fragments in polyacrylamide gel containing a gradient of denaturing agents (denaturing gradient gel electrophoresis). Only fragments with altered melting behavior are sequenced after asymmetric PCR. Beside the frequent polymorphism already described on Pro 626, we detected 18 different sequence variations by studying exons II, IV, V, VIII, X, and XV in 19 of 100 consecutive patients with protein S deficiency. Fifteen were candidate causal mutations, 4 of which were associated with a qualitative deficiency (type IIa or IIb). The remaining three sequence variations were probably polymorphisms.