Six 1-3H-labeled analogues of farnesyl pyrophosphate have been studied as potential substrates for yeast and rat liver squalene synthetases: 2-methylfarnesyl pyrophosphate (4), 3-demethylfarnesyl pyrophosphate (5), 7,11-dimethyl-3-ethyl-2,6,10-dodecatrienyl pyrophosphate (6), 6,7,10,11-tetrahydrofarnesyl pyrophosphate (7), 4-methylthiofarnesyl pyrophosphate (8), and 4-fluorofarnesyl pyrophosphate (9). Analogues 4 and 5 are enzymatically incorporated into 11-methylsqualene (10) and 10-demethylsqualene (11), respectively, even if no farnesyl pyrophosphate is added to the incubations. None of the other analogues gives nonpolar products with either the yeast or liver enzymes. No tritium is enzymatically released to the medium from any of the analogues, indicating that they are not accepted at the first (proton exchanging) site. The data rule out formation of dead-end presqualene pyrophosphate products with analogues as first, but not as second, substrates. Implications of these results for the enzyme active-site topology and mechanism are discussed.