To identify genes involved in the formation of genetic tumors in interspecific hybrids (F1) between Nicotiana glauca and N. langsdorffii, genetic tumor-related cDNA probes were obtained by a subtractive hybridization procedure and used to screen libraries of genetic tumor cDNAs. As a result, 17 distinct cDNA clones were isolated for genes that are specifically or preferentially expressed in genetic tumor tissues but are not expressed at all or are barely expressed in normal F1 plants. Among the isolated clones were genes that encoded so-called stress proteins, such as glucan endo-1,3-beta-glucosidase, osmotin, pathogenesis-related proteins and proteinase inhibitor I. Transcripts corresponding to two of the isolated cDNA clones accumulated to a significant extent only in genetic tumor tissues and were not present in callus tissues from parental plants or in the stems and leaves from normal F1 plants. Analysis of genomic DNA revealed that four of these clones hybridized only to genomic sequences from N. langsdorffii and one hybridized only to a genomic sequence from N. glauca. Eight apparently novel clones were further analyzed to determine the kinetics of accumulation of the corresponding mRNAs during development of genetic tumors. The patterns of accumulation of the mRNAs after induction of tumors by cutting of F1 stems could be divided into three groups, an indication that at least three distinct regulatory mechanisms are operative at the transcriptional level to control the expression of these tumor-related genes during the formation of genetic tumors.