To establish a protocol for studying effects of pulmonary exposure in developmental toxicity studies, the effects of intratracheal sham instillation under short-term isoflurane anaesthesia were evaluated with a protocol including multiple instillations during gestation. Twelve time-mated mice (C57BL/6BomTac) were anaesthetized with isoflurane and intratracheally instilled with saline containing 10% bronchoalveolar lavage (BAL) on gestation days 8, 11, 15 and 18. In addition, the early effects of the procedure were assessed in naive female mice. Control animals were not handled. Dams were followed until weaning, and the offspring were observed from birth to sexual maturation. The cell composition of BAL was examined in the females early after treatment (3 days) and in the dams at weaning (25 days). DNA damage in BAL and liver cells was determined by the comet assay. The procedure did not affect gestation or viability, growth and sexual maturation of the offspring. Lung markers of inflammation and DNA damage were comparable in control and treated dams. Livers of the anaesthetized and instilled females, dams and their offspring displayed no induction of DNA damage. Intratracheal instillation under isoflurane anaesthesia did not induce observable effects in pregnant mice or their offspring. We suggest that this procedure can be used as a means of exposure through the airways in studies of developmental toxicity.