A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-beta-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-beta-D-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, gamma = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 micrograms of crude fibroblast cytosol protein.