Maternal heat stress during the dry period affects calf performance during postnatal life. The objective was to evaluate whether calves that experienced heat stress in utero have altered thermoregulatory responses to acute heat stress later in life. Cows used in the study were born to dams exposed to heat stress (HT) or cooled (CL) during the dry period preceding their birth. All animals were raised postnatally under identical management. Eight HT (173 ± 132 DIM; 28.9 ± 5.2 kg/d milk) and 8 CL (167 ± 124 DIM; 30.1 ± 8.0 kg/d milk) lactating Holstein cows were used for the study. A heat stress challenge was conducted in 2 blocks using 4 HT and 4 CL cows matched according to milk yield, stage of lactation, and parity. Each challenge, which consisted of transfer from a barn with shade and evaporative cooling to one with shade but no additional cooling for a period of 48 h, was replicated twice for each block. Sweating rate, respiration rate, rectal temperature (RT), and skin surface temperature were measured on each cow at 0900, 1100, 1300, 1500 and 1700 h for 2 consecutive days. Mean ambient temperature across 4 challenge days was 23.8 ± 3.8°C. Sweating rate and skin temperature were measured on the right side of the cow on both shaved (5 × 5 cm) and un-shaved areas on the rump of the cow. During the challenge, differences were observed between treatments for RT (CL: 39.2 ± 0.06; HT: 39.0 ± 0.06°C; P = 0.02) and respiration rate (CL: 68.1 ± 1.6; HT: 62.2 ± 1.6 breath/min; P = 0.01). There were also tendencies for differences in sweating rate for shaved skin (CL: 30.7 ± 1.6; HT: 27.1 ± 1.6 g/m²h; P = 0.12) and for unshaved skin (CL: 23.95 ± 1.18; HT: 21.44 ± 1.18 g/m²h; P = 0.13). There was no effect on skin temperature at the shaved area (CL: 35.28 ± 0.12; HT: 35.32 ± 0.12°C; P = 0.81) or the un-shaved area (CL: 34.1 ± 0.14; HT: 34.0 ± 0.14°C; P = 0.55). The results support the hypothesis that heat stress in utero in late gestation increases heat tolerance at maturity by increasing capacity to dissipate heat to maintain core body temperature.