Background: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis.: Objectives: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. Subjects: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. Methods: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247-279, 509-528, and 524-543, proinsulin a.a. 9-23, and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853-872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, tumor necrosis factor β, transforming growth factor β1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. Results: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-γ (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. Conclusion: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy. © 2007 The Authors Journal compilation © 2007 Blackwell Munksgaard.