The interaction of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) with peroxin Pex21p was identified in a two-hybrid screen with SerRS as bait. This was confirmed by an in vitro binding assay with truncated Pex21p fused to glutathione S-transferase. Furthermore, purified Pex21p acts as an activator of yeast seryl-tRNA synthetase in aminoacylation in vitro, revealing the functional significance of the Pex21p-SerRS interaction. Pex21p is a protein involved in the peroxisome biogenesis [Purdue, P.E., Yang, X. and Lazarow, P.B., J. Cell Biol. 143 (1998) 1859-1869]. Since eukaryotic aminoacyl-tRNA synthetases are known to participate in assembles with other synthetases and non-synthetase proteins, we propose that this unusual interaction reflects another function of the peroxin.