An endoplasmic-reticulum — DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2′,3′-dideoxyribosylthymine 5′-triphosphate (ddTTP) and α-amanitin, suggesting the involvement of DNA polymerase α. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 μg/ml α-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [γ-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7–11 bases in length) at the 5′ ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1–90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60–200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100–6000 bases) were synthesized with DNA polymerase α solubilized from the endoplasmic-reticulum — DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.