Raised T-cell proliferation of cord blood mononuclear cells (CBMC) in response to various ingestant and inhalant allergens has been reported in newborns, suggesting a prenatal allergen contact. In general, for in vitro proliferation assays a concentration of 50 x 103 or 100 x 103 cells/well are used. The aim of this study was to analyze whether cell concentration influences T-cell reactivity in cord blood cells and to study differences of T-cell reactivity triggered by inhalant and ingestant allergens. CBMC from 51 neonates (34 females: 22 with and 29 without a family history of allergy, i.e. FH+ or FH-) were incubated with interleukin-2 (IL-2), β-lactoglobulin (β-LG), ovalbumin (OVA), house dust mite allergen Dermatophagoides pteronyssinus (Der p 1), and timothy grass allergen Phleum pratense (Phl p 1) for 7 days. The cell concentration ranged from 6.25 x 103 to 100 x 103 cells/well. Proliferation was assessed by incorporation of [3H]-thymidine and was expressed as counts per minute (c.p.m.). In unstimulated cells, a decreasing cell concentration paralleled a steep drop of background activity. In response to IL-2, a decreasing cell concentration led to a slow decrease of c.p.m. The corresponding mean stimulation indices (SI) were 9, 32, 77, 47, and 21 for 100 x 103, 50 x 103, 25 x 103 12.5 x 103, and 6.25 x 103 cells/well, respectively. In addition, the highest number of positive proliferative responses to specific allergens were obscured at lower cell concentrations. For β-LG, the maximal number of positive responses were obtained between 25 x 103 (n = 44) and 12.5 X 103 (n = 46) cells/well, for OVA at 25 x 103 (n = 3) cells/well, for Der p 1 at 50 x 103 (n = 5) cells/well, and for Phl p 1 between 25 x 103 and 12.5 x 103 (n = 5) cells/well. Positive proliferation in at least one of the tested assays was observed in 100% of samples in response to β-LG, in 22% in response to Phl p 1, and in 14% in response to OVA and Der p 1. T-cell reactivity did not differ between samples of newborns with or without a family history of atopy. Therefore, sensitivity of T-cell proliferation measurement is highly influenced by background proliferation of unstimulated cells. Hence, proliferation assays with lower cell numbers unmask T-cell reactivity in response to ingestant and inhalant allergens. We suggest the use of concentrations of 12.5 x 103-50 x 103 cells/well in proliferation experiments.