Previous studies of the toxicity of candidate fuel additives identified severe testicular toxicity in animals exposed to 1,6-dimethoxyhexane (DMH). A series of studies were conducted to characterize the effects of DMH on spermatogenesis and to compare the effects of DMH with responses to structural similar aliphatic ethers. In the first study, sexually mature male rats were administered a single dose (600 mg/kg) of DMH, and subsets of animals were sampled at intervals post exposure (PE). Both testis and thymus weight declined steadily after DMH exposure, both being significantly lower than control by 7 days PE. Treatment with DMH led, at 24 to 48 h PE, to an increase in dying primary spermatocytes in seminiferous tubule stages I-IV and stages XII-XIV, but not intervening stages. The affected cohort of germ cells was seen progressing through the developmental sequence of spermatogenesis as numbers of dying cells returned to control levels by 7 days PE, coincident with a significant decline in the proportion of round spermatids among germ cells as determined by flow cytometry. Resolution of round spermatids to control levels by day 21 PE coincided with a reduction in condensed spermatids (homogenization-resistant spermatid nuclei) and was followed at 28 days PE by a significant reduction in cauda epididymal sperm counts. Further studies of repeated exposures to DMH (200 mg.kg(-1).day(-1), 5 days per week for 4 weeks) confirmed the significant testis toxicity of this material. In contrast, similar treatment with any of a variety of structurally similar aliphatic ethers had little or no impact on testis function. Methoxyacetic acid, previously shown to cause rapid death of some meiotic germ cell stages, was found at high concentrations in the urine of DMH-treated rats but not in rats treated with other ethers, suggesting that DMH exerts its testis toxicity via metabolism to this substance. These results demonstrate that DMH selectively deletes germ cells from the testis At the very early or very late pachytene, diplotene, or M-phase spermatocyte stages, possibly through conversion to MAA.