Photoinitiators used in food packaging ink, such as 2-isopropylthioxanthone (2-ITX), have been shown to migrate into food and beverages. Recently, several studies indicated that 2-ITX might be an endocrine disrupting chemical. In the present work, the effects of 2-ITX, 4-isopropylthioxanthone (4-ITX), 2,4-diethylthioxanthone (2,4-diethyl-TX), 2-chlorothioxanthone (2-chloro-TX) and 1-chloro-4-propoxythioxanthone (1-chloro-4-propoxy-TX) on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. None of the compounds showed androgenic or estrogenic activities, but clear anti-androgenic as well as anti-estrogenic activities were observed for 2-ITX, 4-ITX and 2,4-diethyl-TX, while 2-chloro-TX showed only anti-androgenic activity. In an adapted version of the H295R steroidogenesis assay, using gas chromatography-tandem mass spectrometry analysis of H295R media, all five compounds increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), were significantly up-regulated after incubation of H295R cells with 2-ITX, 4-ITX and 2,4-diethyl-TX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays. The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signalling and steroid hormone production.