Beef insulin as well as a chymotryptic A-chain fragment [BI-A1-14(SSO3-)3] need uptake by antigen-presenting cells (APC) for efficient presentation in combination with major histocompatibility complex class II molecules to insulin-specific T cells. This could be shown by the inability of aldehyde-fixed APC to present these antigens to T cells. Furthermore, presentation of the insulin fragment as well as presentation of ovalbumin (OVA) was inhibited by treatment of APC with chloroquine, cerulenin or tunicamycin. This was not the case for a processing-independent OVA peptide. Treatment of APC during antigen pulsing with various protease inhibitors, active on all classes of proteases, did not block presentation of insulin although some of these reagents did interfere with the presentation of OVA. Several inhibitors especially of serine or thiol proteases rather enhanced the presentation of insulin. This indicates that intracellular proteolytic cleavage of insulin does not seem to be required for generation of the antigenic determinant but, if it occurs, rather destroys the antigenic peptide. Insulin and its A-chain fragment may, therefore, represent a model for a processing-dependent antigen not requiring proteolytic cleavage but other modifications.