The folylpolyglutamate synthetase activities of Neurospora crassa wild type (FGSC 853) and two polyglutamate-deficient mutants (met-6, 35809, FGSC 1330 and mac, 65108, FGSC 3609) were examined using dialyzed extracts prepared during exponential mycelial growth. Enzyme assay was based on incorporation of [U-3H]glutamate in folylpolyglutamates that were separated by gradient elution from DEAE-cellulose. Extracts of the wild type produced H4PteGlu2 (15%), H4PteGlu3 (35%) and H4PteGlu6 (50%) when anaerobically incubated with glutamate, ATP, and H4PteGlu. Under these conditions, the met-6 produced only H4PteGlu2 and higher polyglutamates (H4PteGlu4 and H4PteGlu5) were not utilized. The mac mutant failed to catalyze addition of glutamate to H4PteGlu. However, H4PteGlu2 was effectively converted to the tri-, and hexaglutamates. Mixing wild type and met-6 protein stimulated the formation of tri-, and hexaglutamates. Mixing mac and met-6 extracts resulted in H4PteGlu3 and H4PteGlu6 labeling when glutamate and H4PteGlu were provided. Fractionation of wild type extracts by addition of (NH4)2SO4 or by differential centrifugation provided evidence for different synthetase activities. Protein of the 0-35% (NH4)2SO4 fraction and that associated with the mitochondrial pellet, catalyzed an H4PteGlu2 leads to H4PteGlu3 reaction. These fractions failed to utilize H4PteGlu or the corresponding tetra-, and pentagluatmates. This triglutamate-forming activity was lacking in mac and met-6. The 45-60% (NH4)2SO4 fraction of the wild type catalyzed formation of di-, tri-, and hexaglutamate from H4PteGlu and glutamate. Hexaglutamate was also formed when the folate substrate wa H4PteGlu2, H2PteGlu4, or H4PteGlu5. These activities were associated with the cytosolic fraction when crude isotonic extracts were centrifuged to remove mitochondria. The characteristic synthetase activities of met-6 and mac were associated with protein of the 45-60% (NH4)2SO4 and cytosolic fractions. It is suggested that folypolyglutamate synthesis in N. crassa involves more than one synthetase-catalyzed reaction. Production of cellular folylhexaglutamate appears to involve two steps, catalyzed by cytosolic enzymes; viz: H4PteGlu leads to H2PteGlu2 followed by H4PteGlu2 leads to H4PteGlu6. These partial reactions are lacking in mac and met-6 respectively. The mitochondrial synthetase of the wild type may not represent a mandatory step in the biosynthesis of folylhexaglutamate but could have significance in generation of compartmented folylpolyglutamates.