Wnt signaling has recently been implicated in the pathogenesis of cancer. We studied the activity of Wnt signaling in peripheral blood CLL lymphocytes, and the methylation status of seven soluble Wnt antagonists including WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 and SFRP5 by methylation-specific PCR in the peripheral blood CLL lymphocytes and bone marrow samples of CLL patients at diagnosis. In the peripheral blood CLL lymphocytes, constitutive activation of Wnt signaling was detected, associated with hypermethylation of the soluble Wnt inhibitors. In the diagnostic CLL marrow samples, methylation of the seven genes was detected in up to 36.4%. Moreover, twenty-three (52.3%) patients had methylation of at least one of these 7 genes, of whom 14 (60.8%) had methylation of two or more Wnt inhibitors. Apart from an association of advanced age with DKK3 methylation, there was no association of gene hypermethylation with either clinical characteristics (including age, gender, lymphocyte count at diagnosis, Rai stage and poor-risk karyotype) or survival. In conclusion, Wnt signaling is constitutively activated in CLL B-lymphocytes in association with methylation of multiple soluble Wnt antagonists. Methylation of these soluble Wnt antagonists, occasionally multiple genes, in primary CLL marrow samples suggested an important role in CLL pathogenesis. Moreover, our study underscored the importance of studying methylation of a panel of but not individual genes regulating a cellular pathway.