Heat stress affects the performance of poultry species and also induces immunosuppression. Chickens can be treated by thermal conditioning to have better heat stress tolerance. Our purpose was to determine the effect of acute heat stress on the immune response, i.e. antibody production against Newcastle disease virus (NDV) and change in the proportion of leukocyte components, in chicks subjected to prenatal heat conditioning. Eighty as-hatched broiler chicks from the same parent stock were used: control (40 chicks incubated at 36.7 °C from days 18–20 of embryonic life) and thermally manipulated (TM) (40 chicks incubated at 38.4 °C from day 18–20 of embryonic life; 4 h/day). The chickens were exposed to heat stress: at day 19 (31 °C/8 h) and at day 35 (32 °C/10 h). The first heat stress (day 19) decreased the lymphocyte counts and significantly increased the heterophil counts (P < 0.05) in both treatments (from 34.25 to 55% in the controls and from 37 to 60.06% in the TM chicks). The second heat stress (day 35) did not alter the heterophil and lymphocyte profiles of the chickens. Before the heat stress, all of the chickens (control and TM) presented the same positive antibody titres to NDV vaccination. After the first heat stress, 50% of the control samples and 40% of samples from the TM chickens were negative. After the second heat stress, 80% of the TM samples were negative.