1. The subcellular distribution of endogenous folate and of injected [3H]folic acid has been studied in guinea-pig liver.

2. Endogenous folate was found to be concentrated in the mitochondrial and cell-sap fractions.

3. At 1 h after injection, labelled folic acid (pteroylglutamic acid) was concentrated in the microsomes (where it is possibly reduced to tetrahydrofolic acid).

4. At all subsequent times after injection of labelled pteroylglutamic acid (4, 24 and 84 h) radioactivity was concentrated in mitochondria and cell sap and it is, therefore, likely that conjugation of glutamates to form pteroylpolyglutamates occurs in one or both of these subcellular fractions in mammalian cells.

5. Methotrexate-treated animals converted at least some labelled pteroylglutamic acid into non-reduced pteroyl di-, tri- and poly-glutamic acids by 4 h after the injection.

6. Reduction of pteroylglutamic acid is, therefore, not essential for pteroylpolyglutamate formation in liver cells.

7. Methotrexate blocks the reduction of pteroyl di-, tri- and poly-glutamic acids, as well as of pteroylglutamic acid.

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