A method for the isolation of polyoma nucleoprotein complexes has been developed using neuraminidase treatment of infected cell lysates. At least three distinct forms of polyoma virion intermediates were identified by their [3H]thymidine labeling kinetics and sedimentation coefficients: a rapidly labeled 95 S "replicating complex" which chases to a 75 S minichromosome and then to a 240 S virion structure. The general properties of these distinct intermediates were similar to those found for SV40. In contrast to SV40, however, a continuum of labeled polyoma viral DNA sedimented between 240 S and 95 S. These complexes were characterized by their release from cell debris with neuraminidase, precipitation with antivirion antibody, complete disruption in 1 M NaCl, and association with hemagglutinating (HA) activity. These intermediates may represent incremental capsid protein additions to the 75 S minichromosome, hypothesized in the current models for SV40 assembly. The ability to isolate a complete complement of polyoma subviral complexes provides a basis for studying the growth defect of polyoma host-range mutants, and the properties of neuraminidase release, hemagglutination, and specific immunoprecipitation suggest purification steps for further characterization of these virion assembly intermediates.