Human leukocyte antigen (HLA) class I and class II molecules differ with respect to their intracellular pathways and the compartments where they associate with processed antigen. To study possible consequences of these differences for the kinetics of antigen presentation by HLA class I and class II molecules, we analyzed changes in the concentrations of free intracellular calcium ions in influenza virus-specific T cell clones after recognition of specific antigen/HLA complexes. HLA class II-restricted viral antigen presentation by Epstein-Barr virus-transformed B lymphoblastoid cell lines (B-LCL) to CD4+ T cell clones started within 1 h and showed little variability, irrespective of antigen specificity or restriction element tested. In contrast, kinetics of viral antigen presentation by HLA class I molecules to CD8+ T cell clones were slower and differed for three antigen/HLA class I complexes tested. While B-LCL presented antigen by HLA-A2 and by HLA-B37 after at least 2 h, they only started to present antigen in the context of HLA-B7 after more than 4 h. This difference in kinetics did not correlate with differences in bulk transport rates of HLA-A2, HLA-B37, and HLA-B7, but seemed greatly influenced by differential rates of peptide generation. Brefeldin A treatment of B-LCL showed for both HLA class I and class II that de novo synthesized HLA molecules were involved in antigen presentation. Thus, differences between intracellular pathways of HLA class I and class II molecules may result in different kinetics of antigen presentation.