Cluster of differentiation molecule (CD)3 and CD28 receptors play crucial roles in T-lymphocyte proliferation. Fe deficiency in man and animals impairs T-lymphocyte proliferation by unknown mechanisms. To test the hypothesis that reduced CD3 and CD28 expression is one of them, thymocytes and splenocytes from control (C; n 24), Fe-deficient (ID; n 24), pair-fed (PF; n 24), and ID mice that were Fe-repleted for 3 (R3; n 24) or 14d (R14; n 12) were labelled with anti-CD3-fluorescein isothiocyanate and anti-CD28-phycoerythrin antibodies. Positive cells were analysed by flow cytometry. Significant differences were observed among groups in the mean levels of haemoglobin and liver Fe stores (C=PF=R14>R3>ID; P<0·005). While Fe deficiency slightly increased the percentage of CD3+ splenocytes, it reduced that of CD28+ thymocytes in mice with thymus atrophy and splenomegaly (P<0·05). These changes were corrected by Fe repletion. CD28 mean fluorescence intensity (FI) was lower and CD3 FI was higher in lymphocytes from R3 and ID, especially those with splenomegaly, than in those from R14 and PF mice (P<0·05). In vitro Fe chelation by deferoxamine (60min) significantly decreased CD28 expression (P<0·05), and slightly increased that of CD3 (P>0·05). Spleen cell proliferative responses to concanavalin A and anti-CD3±anti-CD28 were reduced by Fe deficiency (ID≤R3<C=PF<R14; P<0·05); and they correlated with FI and percentages of CD3+ and CD28+ cells (r≤0·69; P<0·05). Indicators of Fe status negatively correlated with CD3 FI (r−0·23), but positively correlated with CD28 FI (r≤0·44; P<0·05). Data suggest that altered CD28 expression may contribute to reduced T-cell proliferation during Fe deficiency.