Many of the proteins in the chloroplast envelope play an important role in facilitating the biochemical and transport processes of the compartment. For the transport of proteins into the chloroplast, we have recently identified at least three different envelope proteins (Com44/Cim44, Com70, and Cim97) in close physical proximity to a partially translocated chimeric precursor protein (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264 -32271). In this study we report the characterization of a cDNA clone encoding a member of the Com44/Cim44 envelope proteins. The combined data from nucleotide sequencing, and RNA and protein blot analyses indicate the existence of multiple forms of the 44-kDa envelope protein. Depending on the plant species examined, immunologically-related protein bands with molecular masses of 42 to 46 kDa were observed. Organelle subfractionation, protease treatment, and immunomicroscopic studies together provide an indication that the immunologically-related proteins may be present in both the outer and inner envelope membranes. Co-migration of the product synthesized from the cDNA insert with a 44-kDa immunoreactive band of the chloroplast envelope, and the in vitro import results, together suggest that the in vitro synthesized 44-kDa protein is targeted to the envelope membrane without any further processing.Chloroplast envelope proteins play a major role in modulating the vectorial flow of molecules across the membrane, including large proteinaceous entities. The import of proteins into the chloroplast is a complex process requiring the close collaboration of both the outer envelope and the inner envelope membranes. Evidence for the possible existence of two distinct protein import complexes, one in each envelope membrane, is beginning to emerge from a number of recent investigations (Waegemann and Soll, 1991;Soll and Waegemann, 1992;Schnell and Blobel, 1993;Alefson et al., 1994; Schell et al., 1994;Kessler et al., 1994;Wu et al., 1994). An important step in the characterization of the protein translocating complexes is the identification of the components involved. The identification of outer and inner envelope polypeptides of these protein translocating complexes has been achieved using a variety of strategies (Cornwall and Keegstra, 1987;Kaderbhai et al., 1988;Pain et al., 1988; Schnell et al., 1990aSchnell et al., , 1994Hinz and Flugge, 1988;Soll and Waegemann, 1992;Waegemann et al., 1990;Perry and Keegstra, 1994;Alefson et al., 1994;Wu et al., 1994;Hirsch et al., 1994;Gray and Row, 1995). So far these studies collectively indicate that envelope proteins with molecular masses of 30, 34, 36, 44, 45, 51, 66, 70, 75, 86, 97, and 100 kDa may be possible constituents of the chloroplast protein import apparatus; however, it is not obvious from the existing data whether some of the predicted similar sized components are identical to each other.The complex nature of protein translocation mechanisms observed in other membranous systems, such as the mitochondrion and the endoplas...