The effects of microi'jection on Ranapipiens oocytes were determined using cryomicrodissection to measure Na, K, water, and injected radiolabeled sucrose (in gelatin) in the nucleus, animal, and vegetal ooplasm and injected bolus (reference phase, RP). The results point to potential problems in the interpretation of microinjection experiments. When oocytes were injected and incubated in Ringer's solution, nucleus, ooplasm, and RP lost K and sucrose and gained Na. Patterns of loss and gain were complex but were consistent with continuous solute leakage at the injection site causing artifactual intracellular diffusion gradients. In spite of leakage, oocytes completed scheduled meiotic maturation when exposed to progesterone. When oocytes were microin'ected and incubated in paraffin oil (a medium in which polar solutes cannot exchange), nuclear and ooplasmic Na, K, and water concentrations remained identical to those in uninjected cells. Neither microinjection per se nor the injected bolus affected intraoocytic solute distributions. These findings imply that, after microinjection in aqueous media, metabolites are lost from and redistribute in cells, and that these artifactual changes are inadequately reflected in the ability of the cell to carry out a complex process. They also show that injection artifacts can be avoided by injecting and incubating cells under paraffin oil.Because of their large size, amphibian oocytes are often used as cellular "test tubes" into which precursors and effectors are introduced by microinjection. During microinjection, the oocyte membrane is punctured and a bolus of foreign material is introduced. Few studies have addressed the problem of injection damage and resultant artifacts, and those that have only provide estimates of loss of injected material. The fates of endogenous substances seem never to be considered, even though normal cell function clearly depends on maintenance of an optimal intracellular environment.We describe here experiments that distinguish and evaluate separately loss of injected material, changes in endogenous solute concentrations, and alterations in intracellular solute distributions after microinjection. The data show that; after microinjection in aqueous media, constitutive solutes (e.g., metabolites) suffer loss and intracellular redistribution while normally excluded (extracellular) solutes enter the cell. However, our findings also suggest a method to avoid microinjection artifacts: injection and incubation in paraffin oil. Oocytes for oil incubation (2) were rinsed in ion-free isotonic sucrose solution, blotted on filter paper, and transferred to paraffin oil (Fisher), previously washed with Ringer's solution in a separatory funnel. Still in oil, the oocytes were injected as described above. Cells were then transferred to 100 p.l of fresh oil, cooled on ice for 5 min, incubated at 20'C, and frozen. Paraffin oil incubation for up to 2 days does not affect oocyte viability as determined by (i) an unchanged appearance; (ii) the ability to maintain ...