Four preparations of crystalline porcine insulin were fractionated into two peaks by Sephadex gel filtration in 1M acetic acid. The minor peak b was of larger molecular size than the major insulin containing peak c. Peak b was heterogeneous on polyacrylamide disc-gel electrophoresis and after limited trypsin digestion a portion migrated on the gels similar to insulin. Peak b had a high degree of cross-reactivity in a radioimmunoassay for porcine insulin. In comparison with its immunoreactivity, peak b had significantly decreased biologic activity as measured by the rat hemidiaphragm and isolated fat cell assays. The possibility that components of peak b may be present in body fluids requires re-evaluation of the biologic effectiveness of substances detected by insulin radioimmunoassay.