Diagnostic criteria for iron deficiency (ID) and iron deficiency anemia (IDA) in infants are poorly defined. Our aim was to establish appropriate cut-off values for hemoglobin (Hb), plasma ferritin, erythrocyte mean cell volume (MCV), zinc protoporphyrin (ZPP) and soluble transferrin receptors (TfR) in infancy. Exclusively breast-fed infants (n = 263) in Honduras and Sweden were randomly assigned to receive iron supplementation or placebo, and blood samples were obtained at 4, 6 and 9 mo of age. Reference ranges were determined using three different approaches for defining iron-replete infants. The usefulness of several variables for predicting the Hb response to iron was evaluated. We found the following 2 SD cut-off values in iron-replete infants: Hb <105 g/L at 4-6 mo and <100 g/L at 9 mo; ZPP >75 micro mol/mol heme at 4-6 mo and >90 micro mol/mol heme at 9 mo; ferritin <20 micro g/L at 4 mo, <9 micro g/L at 6 mo and <5 micro g/L at 9 mo; and TfR >11 mg/L at 4-9 mo. The Hb response to iron was not a useful definition of IDA at 4 mo of age. Hb, MCV and ZPP at 6 mo as well as growth variables predicted the Hb response at 6-9 mo, but ferritin and TfR at 6 mo did not. We conclude that there is need for a reevaluation of the definitions of ID and IDA in infants.