To evaluate the reliability of phosphorus-31 magnetic resonance (MR) spectroscopy in the assessment of acute testicular ischemia, vascular integrity, and spermatogenesis, the authors studied in vivo canine and primate testicles grouped as follows: group 1 testes (n = 8), in situ canine controls; group 2 (n = 11), canine testes subjected to warm ischemia; group 3, canine (n = 4) and primate (n = 4) testicles from hormone-treated animals. Group 1 control testicles showed high monophosphoester (MP) levels; low levels of inorganic phosphate (Pi), phosphodiester (PD), and phosphocreatine; and high levels of adenosine triphosphate (ATP). Group 2 testes revealed a time-dependent decay of MP/Pi ratios (from 2.1 to 0.70). Regeneration of ATP was noted in the acute reperfusion period. After 6 weeks of pituitary gonadotropin suppression, group 3 testes showed a significant decrease (P less than .05) in MP/PD ratios from a control level of 2.6 +/- 0.3 and a decrease in the MP/beta-ATP ratio from 2.4 +/- 0.1 to 1.8 +/- 0.3. P-31 MR spectroscopy appears to be a potential method for noninvasively assessing testicular ischemic injury and the metabolic integrity of spermatogenesis.