The in vitro secretion of immunoreactive somatomedin C/insulin-like growth factor I (IR Sm-C/IGF-I) by two human breast cancer cell lines, MCF-7 and EVSA-T has been studied. IR Sm-C/IGF-I concentration showed a linear increase in serum-free culture media over 72 h of incubation for both cell lines, and a close correlation with cell number (P less than 0.001). To characterize this immunoreactivity, a pool of conditioned media collected after 72 h of incubation was dialyzed overnight against 1 M acetic acid, lyophilized, and gel filtered on a Sephadex G-50 column. Fractions were determined for Sm-C/IGF-I content and for the presence of a specific carrier for Sm-C/IGF-I. Two peaks of Sm-C/IGF-I-like immunoreactivity were evidenced, the first in the high molecular weight region and the second corresponding to the molecular weight of the free peptide. The first peak evidenced also a specific binding ability for radioiodinated Sm-C/IGF-I, suggesting that the activity found in this region could be interpreted as interference of the specific free binding sites in the immunoassay. Analysis of this peak by polyacrylamide gel electrophoresis demonstrated the presence of a specific binding for Sm-C/IGF-I in a molecular weight range between 35,000 and 45,000 Da, which was not modified in reducing conditions. The binding activity was competitively inhibited by addition of cold Sm-C/IGF-I but not by insulin excess.(ABSTRACT TRUNCATED AT 250 WORDS)