Five human PRL-secreting pituitary tumors were tested for the presence of DNA-transforming sequences. After calcium phosphate transfection to NIH-3T3 mouse fibroblast cells, DNA samples derived from two prolactinomas induced foci of morphologically transformed cells which subsequently grew in soft agar. After retransfection of transformant DNA, resulting secondary transformants elicited rapidly growing solid tumors in nude mice. Southern analysis of transformant DNA revealed the integration of Alu-positive human DNA sequences into the mouse fibroblast NIH-3T3 cells, as judged by hybridization to a Blur-8 probe. The Alu signal became increasingly more difficult to detect with the multiple passaging (>20) of transformant cells in culture. Alu polymerase chain reaction (PCR) was, therefore, used to selectively amplify human DNA sequences from the NIH-3T3 rodent background. PCR using a human Alu-specific primer resulted in amplification of an Alu-containing DNA region within these transformants. The transformant DNA did not hybridize to human genomic probes for genes known to evoke focus formation in this assay, including H-ras, K-ras, N-ras, trk, ret, ros, or met. Further identification of the Alu-containing region revealed that it contained sequences from the human hst gene, a member of the fibroblast growth factor family. The presence of human hst was demonstrated by strong hybridization to a 40-mer oligonucleotide probe to the second exon of hst, by amplification of this region with human hst-nested amplimers within the first and second introns, and finally by direct sequencing. Northern analysis showed hst mRNA transcripts (∼3 kilobases) in nude mouse tumors generated from secondary transformants. Using reverse PCR, a 200-basepair cDNA was amplified from prolactinoma tumor RNA, with amplimers bracketing the first and third exons and shown to hybridize to an hst oligonucleotide probe for the second hst exon. A highly sensitive ribonuclease protection assay also confirmed the presence of hst transcripts in a prolactinoma. These results demonstrate that DNA sequences from human prolactinomas containing at least part of the coding region for the hst gene are transforming in the NIH-3T3 cell focus assay. Expression of hst mRNA in prolactinomas suggests that this growth factor gene may be associated with pituitary tumorigenesis.