The herpes simplex virus 1 capsid is formed in the infected cell nucleus by way of a spherical, less robust intermediate called the procapsid, Procapsid assembly requires the capsid shell proteins (VP5, VP1SC, and VP23) plus the scaffolding protein, pre-VP22a, a major component of the procapsid that is not present in the mature virion. Pre-VP22a is lost as DNA is packaged and the procapsid is transformed into the mature, icosahedral capsid. We have employed a cell-free assembly system to examine the role of the scaffolding protein in procapsid formation, While other reaction components (VP5, VP1SC, and VP23) were held constant, the pre-VP22a concentration was varied, and the resulting procapsids were analyzed by electron microscopy and SDS-polyacrylamide gel electrophoresis. The results demonstrated that while standard-sized (T = 16) procapsids with a measured diameter of similar to 100 nm were formed above a threshold pre-VP22a concentration, at lower concentrations procapsids were smaller. The measured diameter was similar to 78 nm and the predicted triangulation number was 9. No procapsids larger than the standard size or smaller than 78-nm procapsids were observed in appreciable numbers at any pre-VP22a concentration tested. SDS-polyacrylamide gel analyses indicated that small procapsids contained a reduced amount of scaffolding protein compared to the standard 100-nm form, The observations indicate that the scaffolding protein concentration affects the structure of nascent procapsids with a minimum amount required for assembly of procapsids with the standard radius of curvature and scaffolding protein content.