The manner by which endocrine-disrupting xenobiotics, such as phthalates, can induce changes in the development of the male reproductive system still remains largely unknown. Herein, we have explored the application of ethane dimethane sulphonate (EDS) to eliminate adult-type Leydig cells in the mature rat testis, leading to their regeneration from resident stem cells, as a novel system to investigate the effects of dibutyl phthalate (DBP) and diethylstilbestrol (DES) on adult-type Leydig cell differentiation. The advantage of this model is that one can study adult-type Leydig cell differentiation in vivo divorced from the concomitant endocrine development of puberty. In these preliminary studies, we show that both DBP and/or DES, given for 2 or 4 days following EDS application, indeed affect Leydig cell differentiation in the adult testis, largely by increasing early Leydig cell proliferation and possibly thereby delaying early differentiation. In particular, on day 27 post-EDS, a time-point when the differentiation trajectory appears to be most discriminating, we observe that both DBP and/or DES cause a fourfold increase in Leydig cell density, and a significant increase in the expression of the Leydig cell-specific marker transcripts INSL3, LH receptor, Cyp17a1 and Cyp 11a1. In conclusion, both DBP and DES are able to affect adult-type Leydig cells during their differentiation to cause a significant perturbation in their ultimate functional capacity.