OBJECTIVE To develop a simple microbiological assay for serum and red cell folates on 96-well microtitre plates, suitable for use in routine clinical diagnosis. METHODS Use of a chloramphenicol resistant organism (NCIB 10463) saved time by avoiding aseptic precautions. Use of plate sealers facilitated mixing. Evaluation of assay performance included estimations of folate recovery, assay reproducibility, and response to reduced folate. Results obtained on sera (193) and red cell folates (150) were compared with those obtained using a traditional microbiological assay. RESULTS Good recovery of folic acid added to serum and also good interassay and intra-assay precision were obtained with both serum (CV% of less than 5) and red cell folate pools (CV% of less than 5). Equimolar assay responses were obtained with folic acid, 5-formyltetrahydrofolate (L-form), and 5-methyltetrahydrofolate (L-form). The microassay correlated well with a traditional assay for estimation of folate in both serum (n = 193, r = 0.975) and red cells (n = 150, r = 0.96). CONCLUSIONS This assay is more compact and less time consuming than the traditional assay. It is extremely economical and is easy to perform in a routine clinical laboratory.