We studied the relationship between differentiation, transformation, and uPA production in a system of rat thyroid cells in vitro. The fully differentiated FRTL5 cells did not produce detectable amounts of uPA, even after stimulation with phorbol esters, potent inducers of uPA expression. All the other cell lines (i.e., FRT, cells which have lost the characteristics of the differentiated thyroid cells; 1-5 G and FRA, transformed cells derived from rat thyroid tumors) produced uPA, the 1-5 G line being the highest producer. Also the FRTL line became positive for uPA production after viral transformation (clone KM4). The lack of uPA expression in FRTL5 cells was not due to the presence of inhibitors and these cells did not produce an inactive molecule, as shown by immunoprecipitation with anti-uPA antibody. However, in FRTL5 cells Northern analysis showed the presence of a small amount of uPA-specific mRNA that increased appreciably after phorbol ester stimulation. In conclusion, in our system uPA expression was a property of undifferentiated and transformed cells; in fully differentiated cells uPA expression was switched off by a still unclear mechanism.