Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a well-known antispermatogenic drug, was studied for the first time in pubertal mice to assess its possible effects on spermatogenesis. Male CD1 mice were orally treated on Postnatal Day (PND) 28 with a single dose of LND (100 mg/kg body weight) and sacrificed on PND30, PND42, PND74 and PND123. On PND30 (48 h after dosing), severe testicular effects were evidenced in the treated animals: (a) reduction of the testicular sperm head concentration (approximately 50% of the control value); (b) changes in the spermatogenic cell type distribution (mild decrease of the elongated spermatids and S-phase cells fractions); and (c) morphological alterations of the Sertoli cell cytoplasm and germ cell exfoliation. These changes were recovered in adulthood, on PND74 and PND123. However, no effect on sperm chromatin structure was detected on the epididymal sperm of mature mice by sperm chromatin structure assay, suggesting that LND did not interfere with the process of chromatin reorganization and DNA packaging.