Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in cattle herds. Two procedures for using serum to detect PI cattle were developed and evaluated. BVDV was found to remain viable for 7 days in serum samples stored at room temperature. The tests use cell culture virus isolation (VI) in 96-well microtiter plates, followed by immunostaining of cell monolayers with monoclonal antibodies. One technique, the immunoperoxidase monolayer assay (IPMA), forms a red intracellular precipitate while the other, the monolayer enzyme-linked immunosorbent assay (M-ELISA) produces a yellow color in solution. The optimal incubation period for microtiter VI was determined to be 4 days. Optimal IPMA staining was obtained by fixing cell monolayers with 20 to 30% acetone, whereas a simple dry-rehydrate-dry cycle provided optimal M-ELISA staining. The M-ELISA and IPMA had the same sensitivities and specificities, but the M-ELISA was a more rapid procedure and use of a spectrophotometer for reading samples allowed for greater objectivity. When compared to standard VI with routine samples submitted for the diagnosis of BVD, M-ELISA and IPMA had a relative sensitivity of 85% and a relatively specificity of 100%. When only samples from cattle suspected of being PI were considered, these two parameters were 100% for both IPMA and M-ELISA. The two procedures, especially the M-ELISA, are suitable for whole-herd testing to identify PI cattle. The appeal of these tests is derived from the convenience of using serum as a diagnostic sample and the ability to rapidly screen large numbers of samples at low cost.