Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.