Levels of nonantigen‐induced pro‐inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)‐matched type 1 diabetes mellitus patients, first‐degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro‐ and anti‐inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage‐colony stimulating factor (M‐CSF) to determine lipopolysaccharide (LPS)‐stimulated tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, IL‐12 and prostaglandin E‐2 (PGE‐2) secretion from hetero‐ or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first‐degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS‐stimulated secretion of TNF‐α, IL‐1β and IL‐6 was immediate and markedly higher in the HLA‐DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA‐matched healthy first‐degree relatives. In DQB1*0201/*0302 diabetes patients PGE‐2 secretion was delayed but increased by LPS stimulation compared with HLA‐matched healthy relatives. IL‐12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE‐2 following nonantigenic stimulation. Sensitized macrophages may be important to high‐risk DQB1*0201/*0302‐associated type 1 diabetes.