We show here that expression of the Escherichia coli cat gene in mammalian cells results in accumulation of enzymatically active CAT in the culture media as well as in the cytoplasm. We call the extracellular product secreted CAT (sCAT). Three to four days after introduction of cat-expressing plasmids into mouse L cells by transient transfection, total extracellular sCAT activity exceeds total cytoplasmic CAT activity. As sCAT levels increase, substantially more CAT is found outside the cells than inside at later times. Comparison of different populations of cat-expressing cells shows that, at any given time, the level of sCAT is proportional to the level of intracellular CAT. Thus, assay of sCAT provides a convenient, non-invasive alternative to assay of intracellular CAT. The molecular sizes of sCAT and intracellular CAT are indistinguishable, suggesting that the protein is not cleaved or glycosylated during secretion. Several observations, including a lack of sensitivity to drugs which inhibit Golgi activity, suggest that CAT may be secreted via an unusual pathway.