Although some of the other commonly used intravenous anaesthetics have been shown to be protective in acute lung injury, the effects of midazolam on acute lung injury is not known. We aimed to investigate the effects of midazolam in oleic acid-induced acute lung injury in rats. Thirty animals were randomly allocated to 5 groups: the Control group (n=6); the Solvent group (n=5), to which 0.01 mL g-1 solvent of midazolam was administered intraperitoneally (ip); the Midazolam group (n=7) with 50 mg kg-1 of midazolam given ip; the Oleic acid group (n=6), to which 50 mL of oleic acid was administered intravenously (iv); and the Oleic acid +midazolam group (n=6), which was given 50 mL Oleic acid iv + midazolam 50 mg kg-1 ip. Two histologists who were unaware of the group assignments assessed the acute lung injury via light microscopy (interstitial oedema, alveolar haemorrhage, intraalveolar neutrophils, intraalveolar macrophages and intraalveolar pneumocytes) and electron microscopy (the integrity of the blood-gas barrier, the morphology of the Type I and Type II pneumocytes, and the presence of intraalveolar macrophages and granular material). Midazolam increased the interstitial oedema and total acute lung injury compared to the control group and to the vehicle group. Oleic acid caused significant total lung injury compared to the control group and to the vehicle group. There were no differences between the oleic acid group and the oleic acid+midazolam group regarding the severity of the acute lung injury. Electron microscopic examination further supported the light microscopic findings. We found that oleic acid caused significant acute lung injury and although midazolam increased interstitial oedema and acute lung injury by itself, it did not affect oleic acid induced acute lung injury. We believe further studies about the effects of midazolam on lungs and on acute lung injury are needed.