Islets from male B10.BR mice (H-2(k)) were isolated by the collagenase technique, hand-picked with a Pasteur pipette, and incubated in tissue culture media supplemented with recombinant murine interferon-gamma (rIFN-γ), recombinant murine tumor necrosis factor (rTNF), or both. IA(k)-molecules could be identified on the surface of islets incubated for 5 days with a combination of rIFN-γ (1, 10, or 100 ng/ml) and rTNF (10 or 50 U/ml) by indirect immunofluorescence. Optimal concentrations of rIFN-γ and rTNF, 10 ng/ml and 50 U/ml, respectively, were used in all subsequent experiments. Weak Ia-positivity could be identified on the surface of islets cultured with both cytokines for as little as 48 hours; however, the staining appeared most intense after 5 days of culture. Intensely Ia-positive islets were then carefully washed and cultured in media without either cytokine; Ia positivity could be identified on the surface of these islets for up to 1 week. Dispersed islet cells were cultured with rIFN-γ alone (10 ng/ml), rTNF alone (50 U/ml), or both cytokines for 5 or 10 days. After either 5 or 10 days of culture with both cytokines, intense immunofluorescent staining for Ia could be identified on the surface of greater than 80-90% of the viable islet cells. Culture with IFN alone for 10 days resulted in 15-20% Ia positivity; culture with TNF alone did not cause Ia expression.