The kisspeptin/Gpr54 signalling pathway plays a critical role in reproduction by stimulating the secretion of gonadotrophin‐releasing hormone (GnRH), yet mice carrying mutations in Kiss1 (which encodes kisspeptin) or Gpr54 exhibit partial sexual maturation. For example, a proportion of female Kiss1^−/− and Gpr54^−/− mice exhibit vaginal oestrus, and some male Kiss1^−/− and Gpr54^−/− mice exhibit spermatogenesis. To characterise this partial sexual maturation, we examined the vaginal cytology of female Kiss1^−/− and Gpr54^−/− mice over time. Almost all mutant mice eventually enter oestrus, and then spontaneously transition from oestrus to dioestrus and back to oestrus again. These transitions are not associated with ovulation, and the frequency of these transitions increases with age. The oestrus exhibited by female Kiss1^−/− and Gpr54^−/− mice was disrupted by the administration of the competitive GnRH antagonist acyline, which also resulted in lower uterine weights and, in Kiss1^−/− mice, lower serum follicle‐stimulating hormone (FSH) and luteinising hormone (LH) concentrations. Similarly, male Kiss1^−/− and Gpr54^−/− mice treated with acyline had smaller testicular sizes and an absence of mature sperm. In addition to examining intact Kiss1^−/− and Gpr54^−/− mice, we also assessed the effects of acyline on gonadotrophin concentrations in gonadectomised mice. Gonadectomy resulted in a significant increase in serum FSH concentrations in male Gpr54^−/− and Kiss1^−/− mice. Acyline administration to gonadectomised Kiss1^−/− and Gpr54^−/− male mice lowered serum FSH and LH concentrations significantly. By contrast to males, gonadectomy did not result in significant gonadotrophin changes in female Kiss1^−/− and Gpr54^−/− mice, but acyline administration was followed by a decrease in LH concentrations. These results demonstrate that, although kisspeptin signalling is critical for the high levels of GnRH activity required for normal sexual maturation and for ovulation, Kiss1^−/− and Gpr54^−/− mice retain some degree of GnRH activity. This GnRH activity is sufficient to produce significant effects on vaginal cytology and uterine weights in female mice and on spermatogenesis and testicular weights in male mice.