Using a rapid purification scheme, we have isolated from calf thymus cells two single strand-specific DNA-binding proteins, with apparent molecular masses of 48 and 61 kDa. These proteins remained undetected in earlier studies on DNA-binding proteins from calf thymus because they are extremely sensitive to proteolytic breakdown occurring during purification. The proteins are immunologically and biochemically related. They also share a number of functional properties. Both proteins bind noncooperatively to single-stranded DNA and almost totally ignore double-stranded DNA. Both proteins stimulate DNA synthesis catalyzed by mammalian DNA polymerase alpha in the presence of an excess of "activated" DNA as primer-template. The stimulation factor was between 2 and 100 depending on the protein/DNA ratio. We have analyzed this effect in more detail with specifically primed single-stranded phage fd DNA as template and concluded that the proteins block nonproductive polymerase-binding sites on single-stranded DNA sequences, thereby increasing the probability for an association of polymerase with 3'-OH primer ends.