For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.