Poliovirus is a small, structurally simple virus that can serve as a powerful model system for the elucidation of the basic processes involved in the genetic control of macromolecular synthesis. The physical and biochemical characterization of temperature-sensitive and drug-resistant mutants of this virus can provide insight into the normal sequence of events during the replication and assembly of the wild-type (wt) virus. The specific interference of the infecting virus with the major pathways of macromolecular synthesis in the host cell offers a possible inroad to the exploration of the relevant control systems. This research is divided into two major segments: (1) a temperature-sensitive mutant of poliovirus type 1, tsB9, and a guanidine-resistant mutant, gʳH, were characterized physiologically; (2) a physiological screening procedure that quickly and efficiently reveals the phenotype of a large number of poliovirus mutants in a short period of time was developed and validated. Two guanidine-resistant and twelve temperature-sensitive mutants of poliovirus type 1, Mahoney, were generated and compared with wt, defective-interfering particles, gʳH, and tsB9 using the screening procedure herein developed. The temperature-sensitive mutant, tsB9, appears to be a structural protein mutant bearing a related defect in ribonucleic acid synthesis at the restrictive temperature. The mutant gʳH was found to differ from the wt virus only in the guanidine resistance of its growth and ribonucleic acid synthesis, although a detailed electrophoretic analysis of its proteins was not done. Among the newly isolated mutants, strains were found with defects in each of the major viral functions except one. No mutant was found to be defective in the ability to inhibit host-cell protein synthesis. The screening procedure developed met all of the criteria set for it mentioned above. It has the potential of being adapted to many virus-cell systems for the rapid determination of mutant phenotype.