We have developed a rapid and sensitive microimmunodot blot assay, the antigen spot test (AST), for the detection of bovine coronavirus (BCV) antigen from neonatal calf fecal samples. The AST procedure can be completed in 3.5 h, whereas the previously reported immunodot blot assays require 10 to 12 h. Ninety-six samples can be tested per membrane, and 10 membranes (960 samples) may be processed by a single technologist in 1 working day. The effects of detergents, oxidizing chemicals, chaotropic agents, and enzyme substrates in improving the sensitivity and signal-to-noise ratio of the AST were studied. Finally, the sensitivity and specificity of AST for the detection of BCV antigen were compared to those of a sandwich enzyme-linked immunosorbant assay (ELISA) and a hemagglutination assay (HA). Of 347 field samples tested by all three methods, 94.2% were positive by AST, 91.4% were positive by ELISA, and 86.7% were positive by HA. The sensitivity of the AST was determined to be 100% compared to the results of the ELISA reference method. The specificity of the AST was 67%, which reflects a lower limit of detection of 10⁴ viral particles per ml in a 10% fecal suspension.