As part of our ongoing studies to characterize molecular alterations in a well-defined series of surgically resected esophageal cancers, we examined the expression of 2 ras-regulated genes, whose products (osteopontin and cathepsin L) previously were shown to be associated with tumor invasion and metastasis. RNA was extracted from primary esophageal tumors (adenocarcinomas, 19; squamous-cell carcinomas, 6) and matched histologically normal esophageal mucosa from the distant resection margin. Northern analysis was used to quantitate RNA, relative to an 18S rRNA control, and immunohistochemistry to assess the tissue distribution of osteopontin. In addition, H-, K- and N-ras mutations were studied in the same tissues using PCR and hybridization with allele (mutant)-specific oligonucleotide probes. We demonstrated a K-ras mutation (codon 12, GTT) in one esophageal adenocarcinoma. The ras-regulated gene osteopontin was over-expressed in 100% of squamous-cell carcinomas and in 58% of adenocarcinomas relative to matched normal esophageal mucosa. Patterns of immunoreactivity for osteopontin protein also varied between squamous-cell carcinomas (tumor cell staining) and adenocarcinomas (predominantly tumor-infiltrating macrophages). Expression of cathepsin L also varied with esophageal tumor histology, with over-expression in 58% of primary esophageal adenocarcinomas and 33% of squamous-cell cancers.