Monoclonal antibody B3 (MAb B3) reacts with many epithelial cancers. It recognizes a carbohydrate antigen (Ley) which is expressed in a variety of solid tumors including breast and colon. We have used the Fab portion of MAb B3 and a portion of the constant domain of human IgG1 to make recombinant immunotoxins of different compositions. The toxin component employed is a truncated form of Pseudomonas exotoxin (PE38). The light chain or Fd of the antibody was cloned from hybridoma RNA and fused to PE38. Immunotoxin (IT) was then expressed in Escherichia coli as a fusion protein and refolded with either the Fd or the light chain. We have also made B3(Fab) immunotoxins of different sizes ranging 85-140 kDa, by introducing different portions of the constant domain of human IgG1 at the junction of Fd and PE38 fusion site. We compared the properties of the resulting immunotoxins with existing anti-Ley immunotoxins side by side. All recombinant Fab-immunotoxins made in this study were cytotoxic to antigen-positive cancer cell lines. However, in contrast to the B3(scFv) immunotoxin, the B3(Fab) immunotoxins are very stable, retaining 90% of their activity after 24 h of incubation in human serum albumin at 37 degreesC. A pharmacokinetics study with these immunotoxin molecules showed a longer survival in the circulation of mice compared to the smaller Fv immunotoxins. The smaller size of the Fab immunotoxins compared to B3Lys-PE38 and the increased T1/2 value compared to B3(scFv)-PE38 and B3(dsFv)-PE38 make these recombinant immunotoxins alternative therapeutic agents to treat Ley antigen positive cancers.