Agonism of human peroxisome proliferator-activated nuclear receptor gamma (PPARγ1) was recently observed in 15 of 25 samples of indoor dust extracts at environmentally relevant exposure levels. In this study, an effect-directed analysis approach was used to identify the primary contributors of PPARγ1 activity in the dust extracts. Three dust extracts showing significant PPARγ1 activity were fractionated with normal phase high-performance liquid chromatography (NP-HPLC) and each fraction was tested for PPARγ1 activity. Three dust extracts showed a similar PPARγ1 activity distribution in the NP-HPLC fractions. In most active fractions, fatty acids (FAs), including oleic acid, stearic acid, palmitic acid and myristic acid, were the primary chemicals identified using gas-chromatography mass spectrometry (GC-MS). Chemical measurements of the FAs in house dust extracts revealed a positive and significant correlation with the observed PPARγ1 activity. To test the role of FAs in the activity, a mixture of four FAs was prepared in the ratios measured in the dust samples and tested for activity. The activity of this mixture was 30-50% of the activity observed in the dust extracts, suggesting they were contributing to the observed activity, but also suggesting additional unknown compounds are likely still present in the dust extracts. To tentatively identify sources of FAs in the dust samples, FAs were quantified in human/animal hair, dead skin cells, and cooking oil. FAs were abundant in all samples and our data indicate that all of these may be sources to indoor dust.