Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.