Wnt signaling has recently been implicated in carcinogenesis. We studied the activity of Wnt signaling and the methylation status of WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 and SFRP5 by methylation-specific PCR in myeloma cell lines and primary myeloma samples. Of the four cell lines, Wnt signaling was constitutively activated in LP1 and WL2, correlating with hypermethylation and hence silencing. Moreover, 5-aza-2'-deoxycytidine treatment of these two cell lines showed progressive demethylation of methylated Wnt inhibitors, re-expression of transcripts and downregulation of Wnt signaling. In both LP1 and WL2 cells, multiple Wnts and Fzs were simultaneously expressed. Treatment of WL2, in which SFRP1 was completely methylated, with recombinant secreted Frizzled-related protein 1 (SFRP1) induced downregulation of Wnt signaling and inhibition of proliferation. In primary myeloma samples, 42% patients had methylation of at least one of these seven genes, of which 61.9% had > or = 2 genes methylated. In conclusion, Wnt signaling is constitutively activated in myeloma, associated with methylation silencing of one or multiple soluble Wnt antagonists. An autocrine loop regulating Wnt signaling was demonstrated in the myeloma plasma cells, in which cellular proliferation was efficiently inhibited by recombinant SFRP1. Methylation study of a panel of genes, regulating a cellular pathway instead of isolated genes, is important.