Transient hypothyroidism induced by propyl-2-thiouracyl (PTU) has been shown to block the formation of the postpartum Leydig cell population. In the present study the effects of prenatally induced chronic hypothyroidism on the development of this adult-type Leydig cell population were investigated, using a more physiological approach. Prior to mating, dams were put on an experimental diet consisting of an iodide poor feed supplemented with a low dose of perchlorate to inhibit the uptake of iodide by the thyroid, and with their offspring, were kept on this diet until sacrifice. In pups at day 12 postpartum plasma thyroid stimulating hormone levels were significantly increased by 20-fold, while thyroxine and free tri-iodothyronine levels were severely depressed to around or below the detection level of the assay, confirming a hypothyroid condition. Fetal-type Leydig cell proliferation was not influenced by hypothyroidism, but progenitor-type Leydig cell formation and proliferation appeared to be reduced by 40 to 60% on days 16 and 28 postpartum. This was followed by increased Leydig cell proliferation at later ages, suggesting a possible slower developmental onset of the adult-type Leydig cell population under hypothyroid conditions. Testosterone levels were significantly increased by 2- to- 10-fold in hypothyroid animals between days 21 and 42 postpartum in comparison to the respective age-matched controls. Combined with the decreased presence of 5-reductase, this implicates a reduced production capacity of 5-reduced androgens. In 84-day-old rats, after correction for the changed body weight/testis weight ratio, plasma insulin-like factor 3 levels were 35% lower in the hypothyroid animals, suggestive of a reduction in the seize of the Leydig cell population. This is confirmed by a 37% reduction in the Sertoli cell/Leydig cell ratio in hypothyroid rats. In conclusion, we show that dietary induced hypothyroidism delays, but unlike PTU, does not block the development of the adult-type Leydig cell population.