Many infants with oesophageal atresia and tracheo-oesophageal fistula (OA/TOF) have associated tracheomalacia (TM), which is one of the reasons for respiratory complications after surgical correction of the atresia. OA/TOF was induced in the offspring of pregnant rats by intraperitoneal injection of adriamycin. Fetuses were harvested by caesarean section. The trachea, oesophagus, lungs, and stomach were removed en bloc and stained for cartilage using Alcian blue. The tracheas were examined, photographed, and relevant parameters pertaining to the tracheal cartilage were measured. Exposure to adriamycin resulted in a range of anatomical defects including OA/TOF (47%) and tracheal agenesis (TA) (41%). Adriamycin-treated fetuses were smaller (P < 0.01), yet had longer tracheas (P < 0.001) than control fetuses. The OA/TOF fetuses had more tracheal cartilage rings than controls (P < 0.01), whereas TA fetuses had fewer (P < 0.001). Both OA/TOF and TA fetuses had more malformed tracheal cartilage rings than controls (P < 0.001 and P < 0.05, respectively). Cartilage in the proximal part of the trachea was most frequently and severely affected (P < 0.05). These observations clarify the structural abnormalities of tracheal cartilage that occur in rat fetuses with OA/TOF or TA induced by adriamycin, and may explain the functional disturbances of TM seen in OA/TOF.