We investigated the spreading pattern of runt-related transcription factor-3 (RUNX3) C-phosphate-G (CpG) island (3478 base pairs) methylation in salivary gland adenoid cystic carcinoma. The methylation status of multiple regions within the runt-related transcription factor-3 promoter CpG island (3478 base pairs) was detected by real-time methylation-specific polymerase chain reaction, and the runt-related transcription factor-3 protein was detected with a Western blot in 19 salivary gland adenoid cystic carcinoma samples and the corresponding nonneoplastic salivary glands. The risk ratio between runt-related transcription factor-3 CpG island methylation and salivary gland adenoid cystic carcinoma progression was analyzed by the logistic analysis of variance model. A possible association between runt-related transcription factor-3 methylation, clinicopathologic parameters, and runt-related transcription factor-3 protein was analyzed. Hypermethylation initially occurs the most at the 5' region of runt-related transcription factor-3 CpG island before spreading to the transcription start site. The extent of methylation was found to be the highest in region nos. 1 and 2 among the successive 10 regions, which extend from the 5' region to the transcription start site within the runt-related transcription factor-3 CpG island. The extent of methylation is lowest at the transcription start site, both in salivary gland adenoid cystic carcinoma and in normal salivary glands. No methylation in the transcription start site was found in normal salivary glands. Logistic analysis of variance model indicates that the transcription start site within the runt-related transcription factor-3 promoter CpG island is critical for gene silencing. Western blots revealed that levels of the runt-related transcription factor-3 protein in adenoid cystic carcinoma samples are significantly lower than those in normal salivary glands (P < .001). Methylation of the runt-related transcription factor-3 CpG island spreads the most from 5' region to the transcription start site in adenoid cystic carcinoma tissues, and the transcription start site may be a critical region for the runt-related transcription factor-3 methylation. The spreading pattern of the runt-related transcription factor-3 methylation may play an a role in the progression of adenoid cystic carcinoma.