The diagnosis of thrombophilia caused by protein S deficiency remains difficult. From 2005 to 2010, we documented 135 patients with suspected hereditary protein S deficiency for whom mutational analysis of the PROS1 gene had been performed by direct double-stranded sequencing of the amplified 15 exons including splice sites. Multiplex ligation-dependent probe amplification was performed on 12 of 15 exons in cases with no mutation found but a large deletion in the PROS1 gene was suspected. Mutations were identified in 49 patients, 9 by familial screening. Altogether, 17 new and 11 previously described mutations of PROS1 were identified among the 49 patients. After the exclusion of acquired protein S deficiency due to pregnancy or hormonal contraceptives, there remained only 1 case with protein S activity levels less than 40% that could not be explained by sequence variations or deletions in the examined regions of the PROS1 gene. After the exclusion of conditions associated with acquired protein S deficiency, persistently low protein S activity levels are highly indicative of a genetic alteration in PROS1. We observed a clear correlation between the laboratory phenotype and the type of mutation.