Air and dust samples were collected on two floors of an office building during a double-blind particle intervention study to examine spatial and temporal variability of airborne endotoxin over a period of weeks, and to characterize endotoxin activity and lipopolysaccharide (LPS) content in carpet and chair dust. Air samples were collected on multiple days within and across weeks. Dust samples were collected from carpets and chairs one day per week for three weeks. Endotoxin was measured using a Limulus assay. Dust samples were analyzed for LPS by determination of 3-hydroxy fatty acids (3-OHFAs) using gas chromatography-mass spectrometry. The geometric mean (geometric standard deviation) for 96 indoor air samples was 0.24 (1.6) EU/m3. Significant within-floor spatial variation of airborne endotoxin was found (P < 0.0001, n = 80). Temporal variability of airborne endotoxin was not significant across weeks. Mean (+/- SD) endotoxin levels in carpet dust (59 +/- 9.3 EU/mg dust, n = 12) and in chair dust (38 +/- 7.7 EU/mg dust, n = 10) were significantly different (P < 0.001). Carbon chain length-dependent differences in 3-OHFA levels by dust source and floor were found. Enhanced air filtration did not significantly affect airborne endotoxin (P = 0.62); however, total dust mass and total endotoxin in carpet dust samples increased significantly after enhanced surface cleaning (P < 0.01). These findings suggest that spatial variability, dust source, and surface cleaning may influence building occupant exposures to endotoxin.