The receptor-binding, immunological and biological properties of synthetic-gene human epidermal growth factor (EGF), produced by recombinant techniques in yeast have been compared with those of mouse submaxillary gland EGF and partially purified EGF from human urine and milk. The three forms of human EGF produced parallel concentration curves in radio-receptor assays using AG2804 Simian virus 40-transformed human lung fibroblasts and iodinated mouse or recombinant human EGF. Equivalent receptor-binding activities of urine and milk EGF were equipotent with recombinant human EGF in an homologous radioimmunoassay using recombinant human EGF antiserum with 125I-labelled recombinant human EGF, while none of the preparations was effective in competing for binding of 125I-labelled mouse EGF to mouse EGF antiserum. Urine, milk and recombinant human EGF stimulated protein synthesis and inhibited protein degradation in cultured AG2804 fibroblasts with identical potency. On a weight basis, mouse EGF was equipotent with recombinant human EGF in competitive binding to cell receptors and in effects on protein synthesis or protein degradation in AG2804 fibroblasts. It is concluded that recombinant human EGF is indistinguishable from the natural growth factor from urine or milk and shares similar biological properties with mouse EGF.