A subtractive library screening was performed to identify changes in gene expression that occur during the process of neoplastic transformation of thyroid cells. A cDNA library was constructed from a human thyroid papillary carcinoma cell line (NPA) subtracted with cDNAs from normal thyroid cells (HTC 2). The differential screening of this library lead to the isolation of 39 cDNA clones; six of them showed homology with a recently isolated gene, named HIP, that codes for a protein belonging to a novel class of heparin/heparan sulfate-binding proteins. Northern blot analysis revealed HIP gene overexpression in all of the human thyroid carcinoma cell lines analyzed, as compared to the HTC 2 cells. HIP expression was particularly abundant in the anaplastic carcinoma-derived cell lines. The analysis of surgically removed thyroid tumors showed overexpression of HIP gene in all of the carcinomas, independent of the histotype, although the largest increase in HIP expression was observed in the undifferentiated forms. In contrast, none of the benign adenomas or normal thyroid tissues showed HIP overexpression. To establish the role of HIP overexpression in cell transformation, the NPA cell line was transfected with an eukaryotic expression vector carrying the HIP gene in the antisense orientation. Stable transfectants expressed reduced HIP mRNA levels and showed morphological changes, such as becoming spindle-shaped and growing scattered. The growth rate of the antisense clones was greatly reduced compared to the NPA cells transfected with the backbone vector. Taken together, these results indicate that HIP gene overexpression is associated with thyroid carcinogenesis and strongly suggest its involvement in thyroid cell growth regulation.