A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains aorund 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs. © 1995.