A random selection procedure was used to isolate a temperature-sensitive (ts) mutant of human adenovirus type 4 (Mastadenovirus h 4 tsl) from nitrous acid mutagenized virus stock. The mutant displayed restricted growth at the nonpermissive temperature of 39 °C. Analysis of the mutant grown at 39 °C, by two-dimensional immunoelectrophoretic analysis, showed the mutant to be defective in the expression of the penton base and fibre structural components. The mutant was, however, capable of synthesizing immunologically reactive hexon components. Temperature-shift experiments revealed detectable fibre and penton to be present following shift-down from 39 to 32 °C. Time-sequence analysis of shift-down experiments suggested a possible defect in processing of the components, as indicated by an increase of immunologically detectable penton base. The ability of the mutant to assemble viruslike particles at 39 °C was confirmed by electron microscopy. Though the particles assembled appeared as mature virions, crystalline arrays of packed particles were less in number and somewhat smaller in size than those observed at 32 °C.