A critical question in fiber research is the relative contribution of chemical properties vs physical dimensions to the potential pathogenicity of an inhaled fibrous particle. To address this question, it is essential to obtain fiber samples of discrete lengths for investigation. Recently our laboratory has developed a method, which utilized a dielectrophoretic classifier, to separate fiber fractions of narrowly defined lengths. The objective of the present study was to analyze the effects of fiber length on the ability of macrophages to phagocytize these fibers and to determine the potency of fibers of various lengths to activate nuclear transcription and cytokine production and to elicit cytotoxicity. Glass fibers (JM-100) were separated into five discrete size fractions (lengths of 3, 4, 7, 17, and 33 m). Fibers 7 m long were phagocytized by macrophages in vitro, while fibers 17 m in length were too long to be completely engulfed, resulting in frustrated phagocytosis. There was a clear distinction in the bioactivity and cytotoxicity of fibers too long to be completely engulfed compared to shorter fibers. Glass fiber fractions having 17 m or 33 m lengths exhibited similar cytotoxicity on macrophages in vitro, measured as lactate dehydrogenase release or inhibition of zymosan-stimulated chemiluminescence. However, these long fibers had a toxic potency nearly two orders of magnitude greater than fiber fractions of 3, 4, and 7 m lengths. Bioactivity was measured as the ability of glass fiber fractions to activate the DNA binding of the transcription factor, nuclear factor kappa B (NFkB), to activate the gene promoter for tumor necrosis factor alpha (TNF ), and to increase TNF production by macrophages in vitro. Long fibers (17 m) were significantly more potent bioactivators than short fibers (7 m). This bioactivation was inhibited by N-acetyl-L-cysteine, an antioxidant, indicating that the generation of oxidants contributed to this induction. These results suggest that length plays an important role in the potential pathogenicity of fibrous particles with effects being magnified when fibers are too long to be phagocytized completely.