We reported previously that different MHC class I molecules can compete with each other for cell surface expression in F(1) hybrid and MHC class I transgenic mice. In this study, we show that the competition also occurs in transfected cell lines, and investigate the mechanism. Cell surface expression of an endogenous class I molecule in Chinese hamster ovary (CHO) cells was strongly down-regulated when the mouse K(d) class I H chain was introduced by transfection. The competition occurred only after K(d) protein translation, not at the level of RNA, and localization studies of a CHO class I-GFP fusion showed that the presence of K(d) caused retention of the hamster class I molecule in the endoplasmic reticulum. The competition was not for beta(2)-microglobulin, because a single chain version of K(d) that included mouse beta(2)-microglobulin also had a similar effect. The competition was not for association with TAP and loading with peptide, because a mutant form of the K(d) class I H chain, not able to associate with TAP, caused the same down-regulation of hamster class I expression. Moreover, K(d) expression led to a similar level of competition in TAP2-negative CHO cells. Competition for cell surface expression was also found between different mouse class I H chains in transfected mouse cells, and this competition prevented association of the H chain with beta(2)-microglobulin. These unexpected new findings show that different class I H chains compete with each other at an early stage of the intracellular assembly pathway, independently of beta(2)-microglobulin and peptide.