Tumor-elicited neutrophils (tcPMN) purified from 13762NF mammary adenocarcinoma tumor-bearing rats enhanced metastasis of syngeneic cells when co-injected intrenously; whereas, circulating (cPMN) and phorbol esteractivated (PMA-PMN) neutrophils did not. We hypothesized that differential protein expression was responsible for functional differences between the neutrophil subtypes. Two-dimensional polyacrylamide gel electrophoresis was used to compare neutrophils (cPMN, PMA-PMN) purified from the peripheral blood of healthy, syngeneic nontumor-bearing rats, to tcPMN collected from rats with highly metastatic [clone MTLn3, subclone MTLn3(T44).5] or poorly metastatic (subclone MTLn3(T44).11] tumors growing in the mammary fat pads. Quantitative differences in polypeptide expression were observed between these functionally distinct PMN populations. Compared to cPMN, expression of a M(r) ≃ 38.8 kDa (pl ≃ 8) polypeptide was similar in tcPMN collected from poorly metastatic tumor-bearing rats, higher in PMA-PMN, and further increased in tcPMN from rats with highly metastatic tumors. Expression of two polypeptides, M(r) ≃ 14.1 kDa (pl ≃ 6) and M(r) ≃ 43.3 kDa (pl ≃ 5), was greater in tcPMN from rats with highly metastatic tumors compared to cPMN, PMA-PMN, or tcPMN from rats bearing poorly metastatic tumors. The fatter two polypeptides thus appeared to be specifically increased in tcPMN from rats bearing highly metastatic tumors. Because it was most abundant and displayed the greatest differences between PMN subtypes, the M(r) ≃ 14.1 kDa protein was further analyzed. Tryptic digests followed by internal sequence analyses of resulting peptide fragments revealed that the M(r) ≃ 14.1 kDa contained amino acid sequences that were identical to those of MRP-14, a 14 kDa neutrophil calcium-binding protein belonging to the S-100 protein family of calcium-binding proteins. These results suggest a novel function for MRP-14 and suggest that MRP-14 may represent a marker for distinguishing phenotypically distinct subpopulations of neutrophils, particularly tcPMN with metastasis-enhancing abilities.