Autosomal recessive congenital ichthyoses are a heterogeneous group of disfiguring skin diseases. They are generally characterized by variable scaling and erythroderma, and patients are frequently collodion babies at birth. Autosomal recessive congenital ichthyoses are represented in 25 of our 50 families by a defective keratinocyte transglutaminase (TGK). Pathogenic classification is difficult to assess on clinical grounds for autosomal recessive congenital ichthyoses and impossible for collodion babies. Thus, we have established a rapid TGK assay in situ on frozen skin sections using incorporation of dansyl-cadaverin to assess transglutaminase (TG) activity in combination with immunohistochemistry for TGK protein. Results were compared with TG activity levels measured in cultured differentiating keratinocytes. Sixteen of 26 patients, including a collodion baby, had strongly diminished TG activity in the cell periphery of differentiating keratinocytes and membrane-bound TG activities in vitro, ranging from 2.2 to 281.3 pmol per h mg. Nine of 26 patients, including a collodion baby, showed strong TG activity in the cell periphery of differentiating keratinocytes in situ and membrane-bound TG activities in vitro ranged from 1519 to 10917 pmol per h mg. In one case, TG assay in situ was ambiguous; however, membranous TG activity in vitro was very low at 76.9 pmol/h x mg. Our results demonstrate an excellent correlation of TG assays in vitro and in situ. In addition, we present a novel test with prognostic value for the collodion baby phenotype. This assay allows rapid pathogenic classification of autosomal recessive congenital ichthyoses with only one caveat that in rare ambiguous cases it might be necessary for proper classification to assess membrane-bound TG activity in vitro.