Telomere shortening in human somatic cells and telomere maintenance in most human immortal cell lines and tumours correlate respectively with the absence and presence of telomerase, the enzyme that synthesizes telomeric DNA de novo . However, approximately 30% of in vitro immortalized human cell lines do not express this enzyme and maintain telomeres by an alternative pathway (ALT) that may also operate in some tumours. Human telomerase is a reverse transcriptase comprising minimally an RNA subunit (hTER) and a catalytic protein moiety (hTERT). Normal somatic cells retain expression of hTER but not of hTERT, and can be converted to a telomerase-positive phenotype by ectopic expression of the catalytic protein. We similarly have restored enzymatic activity to those ALT cell lines that retain hTER expression. We also report that in those ALT cells that are hTER negative, reintroduction of both hTER and hTERT is necessary and sufficient for conversion to telomerase positivity. Moreover, transfection of these cells with hTERT in conjunction with hTERs with a mutated template results in the expression of an enzyme with altered specificity. Reconstitution of telomerase activity in ALT cells, particularly an activity capable of synthesizing mutant telomeric DNA, may be exploited for the study of the ALT mechanism and its interaction with the telomerase-dependent pathway, and for assessing the effects of mutant telomeres on cell viability.