In contrast to some published Studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 μ g protein/ml) for 48 h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p < 0.04). Cytotoxicity, as measured by the cellular release of [C-14]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p < 0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT I KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p < 0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1. © 2005 Elsevier Inc. All rights reserved.