Genetic and biochemical studies have shown that DNA polymerase δ (Polδ) is the major replicative Pol in the eukaryotic cell. Its functional form is the holoenzyme composed of Polδ, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). In this paper, we describe an N-terminal truncated form of DNA polymerase δ (ΔN Polδ) from calf thymus. The ΔN Polδ was stimulated as the full-length Polδ by PCNA in a RF-C-independent Polδ assay. However, when tested for holoenzyme function in a RF-C-dependent Polδ assay in the presence of RF-C, ATP and replication protein A (RP-A), the ΔN Polδ behaved differently. First, the ΔN Polδ lacked holoenzyme functions to a great extent. Second, product size analysis and kinetic experiments showed that the holoenzyme containing ΔN Polδ was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Polδ. The present study provides the first evidence that the N-terminal part of the large subunit of Polδ is involved in holoenzyme function.