The origin of the eukaryotic cell remains an unsolved question. Numerous experimental and phylogenetic observations support the symbiotic origin of the modern eukaryotic cell, with its nucleus and (typically) mitochondria. Incorporation of mitochondria has been proposed to precede development of the nucleus, but it is still unclear whether mitochondria were initially part of basal eukaryotes. Data on alanyl-tRNA synthetase from an early eukaryote and other sources are presented and analyzed here. These data are consistent with the notion that mitochondrial genesis did not significantly precede nucleus formation. Moreover, the data raise the possibility that diplomonads are primary amitochondriates that radiated from the eukaryotic lineage before mitochondria became fully integrated as a cellular organelle.