Telomerase activity is present in most malignant tumors and provides a mechanism for unlimited replication of neoplastic cells. Expression of the gene encoding human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene, is associated with telomerase activity, and it is overexpressed in most colorectal carcinomas. In the present study we assayed telomerase activity by the telomeric repeat amplification protocol (TRAP) and used in situ hybridization (ISH) and the reverse transcription polymerase chain reaction (RT-PCR) to study hTERT expression in colorectal carcinomas and adjacent normal tissues. Telomerase activity was found in 30/35 (85.7%) of normal mucosae and 35/35 (100%) of adenocarcinomas, and RT-PCR detected hTERT in 33/35 (94.3%) of the carcinomas. ISH, on the other hand, detected weak but significant expression of hTERT in a significant percentage of lymphocytes infiltrating normal colorectal mucosa. hTERT gene expression was detected in the nuclei of adenocarcinoma cells in 27/35 (77.1%) of the lesions. The results of our comparison of telomerase activity and hTERT gene expression by RT-PCR-based ISH appeared contradictory, but a careful review suggested that the discrepancy was attributable to contamination by infiltrating lymphocytes. Our findings suggest that ISH-based analysis of hTERT gene expression is superior to both TRAP telomerase activity and hTERT mRNA RT-PCR analysis as a means of determining telomerase status during carcinogenesis.