While several studies have documented the presence of plasminogen activator (PA) activity in hen ovarian follicle granulosa and theca tissues, to date it has not been possible to conclusively distinguish between the urokinase (u) and the tissue-type (t) form of the enzyme; this inability is due, in part, to lack of the cloned or characterized chicken tPA gene or gene product. Thus, the present studies were conducted to identify a partial cDNA for chicken tPA and subsequently to evaluate expression of uPA and tPA mRNA in granulosa and theca tissues in vivo and in vitro. Urokinase PA and mRNA levels were highest in prehierarchical-follicle granulosa (3- to 5- and 6- to 8-mm follicles) and theca (6- to 8-mm follicles) tissue compared to hierarchical (9-12 mm through largest preovulatory) follicles. In vitro treatment with a phorbol ester (phorbol 12-myristate, 13-acetate), but not a cAMP analogue (8-bromo-cAMP), significantly increased uPA mRNA levels in both granulosa and theca tissue from the largest and second-largest preovulatory follicles. Of special interest was the finding that levels of uPA mRNA were 10.9-fold higher in atretic compared to morphologically normal 3- to 5-mm follicles. Moreover, 4- to 8-mm-follicle granulosa cells, which spontaneously undergo apoptosis in vitro, demonstrated a rapid increase in uPA mRNA levels after 1 h of incubation (prior to the detection of oligonucleosome formation) while levels in preovulatory-follicle granulosa cells, which do not undergo spontaneous apoptosis, were not altered after 18 h of incubation. By contrast, while tPA mRNA can be identified in granulosa and theca tissues from prehierarchical and preovulatory follicles following polymerase chain reaction amplification, constitutively expressed levels of the transcript were too low to reliably measure by Northern blot analysis. These data indicate that while the chicken expresses a tPA gene that is homologous to the mammalian tPA, uPA is the predominant PA expressed in the hen ovary. In addition, the higher levels of uPA mRNA found in granulosa cells actively undergoing apoptosis and in follicles most susceptible to atresia (4-8 mm) suggest a role for this protease in mediating processes both during the early stages of programmed cell death and in the later stages of follicle atresia and resorption.