Crosslinking reagents were used to interrupt the process of filamentous phage morphogenesis and investigate the orientation in which nascent virions are extruded through the host cell membrane. Infected bacteria with emerging phage particles were crosslinked with glutaraldehyde. Immunoferritin-labeling studies on these emerging phage using anti-A protein IgG suggested that extrusion begins with the C protein end. To confirm this, phage extruding from infected bacteria were frozen using the reversible crosslinker dimethyl 3,3'-dithiobis-propionimidate and fragments of emerging phage were isolated by shearing. Protein analysis of these fragments showed them to be enriched in C protein relative to A protein, as predicted if phage extrusion begins with the C protein end. The production of multiple-length phage particles (polyphage) by nonpermissive bacterial hosts infected with amber mutant phage strains was also studied. Polyphage were produced upon infection with amber mutants in genes III, VI, VII, and IX which code for proteins found at the ends of the mature phage particle. No polyphage were produced by mutants in the other genes tested. Gene III amber mutants produce noninfective polyphage, but those produced by genes VII and IX are infective. Gene VI amber mutants appear to produce unstable, noninfective polyphage particles.