Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear transcription factor that regulates many genes and is involved in extensive biological functions. Accurately determining PPARγ activity in various tissues is important to understanding mechanisms of human physiology and pathophysiology. Thus, we evaluated a PPARγ DNA binding immunoassay using nuclear extracts of spleen and adipose tissue from rats treated with rosiglitazone (20 mg/kg in food, seven days, n = 6) or vehicle (n = 6) and compared the results to mRNA expression of PPARγ target genes--a well-established method to investigate PPARγ activity. In adipose tissue, the PPARγ immunoassay showed that rosiglitazone did not change PPARγ binding, but qPCR analysis showed that expression of two PPARγ target genes, CD36 and liver X receptor-α, were significantly increased. In spleen, the PPARγ immunoassay showed that rosiglitazone decreased PPARγ binding, but qPCR analysis showed no significant change. The different results obtained between PPARγ binding immunoassay and target gene expression suggest that PPARγ immunoassays may not be suitable when used with fresh homogenates of spleen and adipose tissue. Validation of the assay with each individual tissue is recommended.