The yeast ADE3(1-333) gene which encodes a truncated protein containing the N-terminal 5,10-methylene-tetrahydrofolate (THF) dehydrogenase (D)/5,10-methyl-THF cyclohydrolase (C) domain of cytoplasmic trifunctional C1-THF synthase is able to complement all the phenotypes associated with ade3 mutations in vivo. However, expression of the ADE3(1-333) gene in an ade3 strain does not retain any D activity in vitro. Expression in a yeast ade3 strain of the ADE3(1-333) fused to the Escherichia coli lacZ gene or to the yeast SER2 gene allows detection of D and C activities in vitro. These results indicate that the N-terminal D/C domain of C1-THF synthase requires the C-terminal 10-formyl-THF synthetase domain for stable catalytic activity in vitro.