Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein.