Proteins enter the secretory pathway by translocation across the endoplasmic reticulum (ER) membrane. In Saccharomyces cerevisiae, import of proteins into the ER occurs both cotranslationally and posttranslationally. Presumably, the cotranslational targeting to the ER membrane is directed by the signal recognition particle, as demonstrated in other eukaryotic systems. The deletion of a gene, called CER1, inhibits the translocation of proteins that enter the ER posttranslationally, but not those that enter cotranslationally. This translocation defect is more pronounced at lower temperatures. A strain possessing a null mutation of CER1 in combination with a kar2 temperature-sensitive mutation displays synthetic growth defects, whereas overexpression of the ER DnaJ homolog Scj1p suppresses the translocation defect in cer1Delta strains. CER1 is predicted to encode a 100-kDa polypeptide, residing in the ER lumen that is related to the hsp70 family of molecular chaperones.