We report the construction of 34 cDNA libraries from human tissues and cell lines in lambda phage vectors (Charon BS, Λgt11, ΛSHK, or ΛzapII). The cDNA was synthesized from poly A+ RNA, using oligo-dT as a primer, and size-selected before ligation to vector arms. High-complexity cDNA libraries from human tissues and cell lines should be a valuable resource for genome mapping studies and identification of disease genes.