Sequence analysis of the noncoding first exon (exon 1) of the Syk gene demonstrated the presence of a previously cloned CpG island (GenBank #Z 65706). Transient transfection analysis in Daudi cells demonstrated promoter activity (18-fold increase over parental luciferase plasmid) for a 348 bp BstXI-BsrBI fragment containing this island. This region exhibits a high GC content (approximately 75%), contains several SP1 binding sites and a potential initiator sequence, but lacks a strong TATA consensus. Bisulfite sequencing and methylation-specific PCR (MSP) of this region demonstrated that the Syk promoter CpG island was largely unmethylated in B-lineage leukemia cell lines, control peripheral blood cells, human thymocytes and CD3(+) T lymphocytes. However, dense methylation was seen in four T-lineage leukemia cell lines, Jurkat, H9, Molt 3 and HUT 78. MSP screening of leukemia cells from six T-lineage acute lymphoblastic leukemia (ALL) patients demonstrated methylation of the Syk promoter CpG island in one T-lineage ALL patient. Promoter methylation was correlated with reduced to absent expression of Syk mRNA and SYK protein in the T-lineage leukemia cell lines. Treatment of the leukemia lines Ha and Molt 3, with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR) resulted in increased Syk mRNA expression. The presence of a methylated promoter sequence in these T-lineage leukemia cell lines and in one T-lineage patient suggests a potential role for SYK as a tumor suppressor in T-ALL.