Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading — which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM) — and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl−). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.