Gonadotrophin treatment is capable of increasing intratesticular blood flow and improving sperm fertilization potential. A nitric oxide (NO)-mediated mechanism may play a role. This study aimed at evaluating whether the increase in intratesticular blood flow, in response to human menopausal gonadotrophin (hMG) treatment, is accompanied by an increase in seminal plasma NO levels in 20 normogonadotropic males affected by severe oligoasthenoteratozoospermia. Patients received 150 IU of hMG, three times a week for 3 months. Before starting treatment and at month 3 of therapy, levels of NO in the seminal plasma were determined, followed by ultrasound scanning of testis, Doppler evaluation of intratesticular vascular resistance, serum hormone and conventional semen evaluation. Statistical analysis was performed by using Wilcoxon test; the limit of statistical significance was p < 0.05. At the end of treatment, seminal plasma concentration of NO, conventional semen parameters and testicular volume did not increase significantly. Follicle-stimulating hormone serum level significantly increased (p = 0.014) after treatment, whereas luteinizing hormone, testosterone and oestradiol values did not change. The resistance index of both intratesticular arteries significantly decreased (p = 0.012 and 0.005 for right and left testes respectively). The results of this study confirmed the positive effect of exogenous hMG on testicular blood flow, but failed to demonstrate any effect of hMG treatment on NO levels in the seminal plasma in infertile males.