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The 5′ region for the endothelin receptor B (EDNRB) gene is a complex CpG island giving rise to four individual transcripts initiating within the island. Here, for the first time, we analyze the relationship between methylation and gene expression in a CpG island located in the 5′ region of a gene with multiple transcription start sites. The CpG island was unmethylated in normal prostate and bladder tissue, whereas it became methylated in apparently normal colonic epithelium. Tumors derived from these tissues were frequently hypermethylated relative to the respective normal tissues. Analysis of 11 individual CpG sites located throughout the CpG island showed that specific sites with high methylation levels in several tumors were also methylated in normal tissues, suggesting that they might serve as foci for further de novo methylation. This region also had high levels of methylation in several cancer cell lines, and we found that a low methylation level in a small region within the 5′ region correlated with expression of the 5′-most transcript. Interestingly, almost complete methylation 200–1000 bp downstream of the transcriptional start site did not block expression of this transcript. Finally, we show that treatment with 5-aza-2′-deoxycytidine can induce transcriptional activation of the four EDNRB transcripts. Our results show the existence of differential, tissue-dependent methylation at the EDNRB 5′ region, suggest the existence of a spreading mechanism for de novo methylation, starting from methylation hotspots, and show that hypermethylation immediately 3′ to a transcriptional start site does not prevent initiation.