Two types of cDNA for nonspecific lipid transfer protein (nsLTP), identical to sterol carrier protein 2, of rat liver were cloned; one was 787 base pairs (bp) long containing a 429-bp open reading frame of 143 amino acids, with a mass of 15,303 Da (15-kDa protein). The cDNA from the other type was 1966 bp long, including a 1641-bp open reading frame of 547 amino acids, giving a mass of 59,002 Da (60-kDa protein). The deduced primary sequence for the 15-kDa protein was exactly the same as the published sequence of purified nsLTP, except for an extra N-terminal sequence of 20 amino acids, consistent with the finding that nsLTP is synthesized as a larger precursor and processed to a mature form. The sequence for the 60-kDa protein contained, at the 3' end, the full sequence of the 15-kDa protein, a larger precursor to nsLTP. The 15- and 60-kDa proteins, synthesized in vitro from the respective cDNAs, were both immunoprecipitated by rabbit anti-rat liver nsLTP antibody and comigrated in SDS/PAGE with the proteins made in vitro from total liver RNA. These results shed new light on the dispute among several groups of investigators about the crossreactivity of anti-nsLTP antibody with a higher molecular mass, 60-kDa protein. In Northern blot analysis, two major RNA bands, 0.85 and 2.2 kilobases (kb) long, were detected together with two minor bands of 1.6 and 2.9 kb. The 0.85- and 2.2-kb RNAs most likely encode the 15-and 60-kDa proteins, respectively.