The role of macrophages in vertebrate defense reactions is especially important at low temperatures, which commonly affect representatives of ectothermic species. The aim of this work was to compare the effects of in vivo thermal acclimation and in vitro assay temperatures on peritoneal macrophages (PMs) from fish (carp and goldfish) and amphibians (salamanders and toads). The substratum adherence of PMs was undisturbed over the wide range of temperatures experienced by animals in nature. Dehydrogenases and endocytosis were sensitive to temperature fluctuations in a species-specific manner, more or less dependent on the previous thermal history of animals. The MTT reduction, inhibited by low assay temperatures, was slightly improved in PMs from cold-acclimated amphibians but not fishes. Endocytosis by the amphibian PMs, very efficient over the wide range of assay temperatures, was additionally improved by previous in vivo cold exposure. Contrasting effects of thermal acclimation were most evident in the case of endocytic properties of fish PMs; endocytosis by PMs from cold-acclimated fishes was extraordinarily efficient at a temperature just above zero, which almost completely inhibited cells from fishes adapted to warmth.