Since there is a possibility of inhaling the fibers of multi-wall carbon nanotube (MWCNT) without any agglomeration, it is important that the pulmonary toxicity is evaluated by intratracheal instillation without agglomeration. MWCNT suspended in an artificial lung surfactant (ALS) with or without grinding in an agate mortar was instilled once intratracheally to rats to determine whether differences of the effects to pulmonary toxicity by different amounts of agglomerated MWCNT particle. The MWCNT suspension preparation method with grinding was effective at reducing agglomerates and in increasing uniform dispersion of the fibers. The ground MWCNT induced higher LDH levels and neutrophil ratios in the bronchoalveolar lavage fluid (BALF). There were no remarkable responses in rats in the non-ground MWCNT group, with the exception of inflammatory responses in the early phase. Some histopathological findings varied between rats given the ground MWCNT and non-ground MWCNT. A major difference was an MWCNT-laden macrophage infiltration site in the lung, which were in the alveolus in the ground MWCNT group, and in the interstitium in non-ground MWCNT group. Accordingly, the preparation method with grinding is considered to be effective at reducing agglomerates and ensuring uniform dispersion of the fibers. These findings lead us to conclude that the amount of agglomerates in the suspension is an important factor affecting the pulmonary toxicity of MWCNT.