Nickel oxide with two different particle sizes, micron size (NiO) and submicron size (nNiOm), as well as crystalline silica as a positive control and titanium dioxide as a negative control, were intratracheally instilled in rats and the phospholipid concentration and the protein concentration and surface tension of bronchoalveolar lavage fluid (BALF), which are used in surfactant assessment, were measured to see if they could be effective biomarkers in toxicity assessment. The results showed that the NiO instilled group showed no significant difference compared to the control group throughout the observation period. In contrast, a significant difference was found in the nNiOm instilled group compared to the control group throughout the observation period. Moreover, a significant difference was found in the crystalline silica instilled group for each measurement compared to the control group while for the titanium dioxide group, almost no significant difference was found. These results indicate that submicronsized particles of nickel oxide with smaller median diameters potentially have a stronger biological effect than micron size particles. They also indicate that screening can be done by measuring the phospholipid concentration and the protein concentration and surface tension of BALF.