Telomerase activity is primarily determined by transcriptional regulation of the catalytic subunit, human telomerase reverse transcriptase (hTERT). Several mRNA splice variants for hTERT have been identified, but it is not clear if telomerase activity is determined by the absolute or relative levels of full-length (functional) and variant hTERT transcripts. We have developed an SYBR green-based reverse transcription-quantitative polymerase chain reaction assay for the enumeration of the four common hTERT mRNA variants and correlated these with telomerase activity and telomere length in 24 human melanoma cell lines. All except five of the lines expressed four hTERT transcripts, with an overall significant level of co-occurrence between absolute mRNA levels of full-length alpha+/beta+ hTERT and the three splice variants alpha-/beta+, alpha+/beta-, and alpha-/beta-. On average, alpha+/beta+ made up the majority (48.1%) of transcripts, followed by alpha+/beta- (44.6%), alpha-/beta- (4.4%), and alpha-/beta+ (2.9%). Telomerase activity ranged from 1 to 247 relative telomerase activity and correlated most strongly with the absolute amount of alpha+/beta+ (R = 0.791, P = .000004) and the relative amount of alpha+/beta- (R = -0.465, P = .022). This study shows that telomerase activity in melanoma cells is best determined by the absolute expression of full-length hTERT mRNA and indicates a role for the hTERT beta deletion variant in the negative regulation of enzyme activity.