With the use of proteins derived from Escherichia coli cells expressing the v-H-ras gene product as immunogens and an enzyme-linked immunosorbent assay with whole cells for a screening method, 4 BALB/c mouse hybridoma cell lines (rp-12, rp-28, rp-35, and rp-38) were isolated that produced monoclonal antibodies (MoAbs) showing higher reactivity with murine ras gene-activated cell lines than with normal cell lines. All the MoAbs complexed p21ras from the ras gene-activated cell lines in Western immunoblot analysis and demonstrated a binding property of p21ras to guanine nucleotides. The indirect immunofluorescence assay revealed that MoAbs rp-12 and rp-28 stained the murine and human H- or K-ras-activated cell lines, and MoAbs rp-35 and rp-38 not only stained these cell lines but also weakly stained a human N-ras-activated cell line. All these MoAbs stained the murine fibroblast lines with lower intensity, but they did not stain a human fibroblast line. Further, positive reactions with MoAb rp-12 were seen against human melanomas, but there was no reaction against nevi. The rp-12, rp-28, rp-35, and rp-38 antibodies are useful additions to the MoAbs reacting with p21ras reported previously.