It has always been assumed that during development the embryo and fetus depend only on glycolysis for energy generation and that they do not oxidize fatty acids. Recently, however, we found abundant expression and activity of fatty acid oxidation (FAO) enzymes in the human embryo and fetus. In a search for FAO gene expression during development we came across two embryonic differentiation genes: differentiation defective (dif-1) and congested-like trachea (colt) of Caenorhabditis elegans and Drosophila melanogaster, respectively. Earlier studies showed that expression of these two genes is essential during developmental stages with high energy requirements. Both dif-1 and colt encode proteins with sequence similarity to the mitochondrial carnitine acylcarnitine carrier (CACT), which suggests that the DIF-1 and COLT proteins might be functional orthologues of CACT. To investigate this, we expressed both dif-1 and colt in Saccharomyces cerevisiae. Our results show that DIF-1 and COLT can functionally complement a yeast CACT deletion strain and thus function as carnitine acylcarnitine transporters. This finding is well in line with the recent observation that embryos are capable of oxidizing fatty acids and furthermore implies that FAO is essential during early embryonic development when the energy demand is high.