A small scale sandwich enzyme immunoassay for macromolecular antigens using β-D-galactosidase fromEscherichia coliand horseradish peroxidase as labels is described. An antibody IgG-coated glass ball of 1.0 mm in diameter was incubated with antigen in 5 μl and subsequently with affinity-purified Fab'-enzyme conjugate in 5 μl. Using B-D-galactosidase, the detection limits of ferritin, IgE, a-fetoprotein and chorionic gonadotropin were 0.001 amol (600 molecules), 0.03 amol, 0.03 amol and 0.07 amol, respectively. Using peroxidase, the detection limits of ferritin, α-fetoprotein and chorionic gonadotropin were 0.005 amol, 0.1 amol and 0.2 amol, respectively. These detection limits were 3 to 30-fold less than those obtained by the previous sandwich enzyme imnunoassays using antibody IgG-coated polystyrene balls of 3.2 mn in diameter in 150 μl.