A cell-free enzyme extract from Streptococcus faecium UNH 564P has been prepared. The extract incorporates either [2-14C]mevalonic acid (MVA) or [1-14C]isopentenyl pyrophosphate (IPP) into squalene and the carotenoids of the bacterium. ATP and manganese ion were found to be absolute requirements for MVA incorporation by the extract. Only manganese ion was found to be an absolute requirement for IPP incorporation by the extract. Other cofactors including magnesium ion, glutathione, potassium fluoride, NADP, and FAD significantly increase the incorporation of both substrates into the S. faecium terpenoids. Isolation and purification of the radioactive terpenoids from the cell-free system confirmed that the carotenoids of S. faecium are triterpenoids.