Invasive group A streptococcus (GAS) infections cause 1,100 to 1,300 deaths annually in the United States. Diagnosis is made when Streptococcus pyogenes is isolated from pus or body fluids; however, cultures are not always obtained, and antibiotic treatment can preclude bacterial growth. An immunohistochemical assay for GAS was applied to formalin-fixed tissue samples from 122 patients with suspect GAS infection. Immunohistochemical staining of well-defined cocci and small, granular antigen fragments was observed in 27 cases. S pyogenes was isolated in 18 cases, whereas in 8 cases, immunohistochemical staining was confirmed by amplification of the sepB gene of S pyogenes from paraffin-embedded samples in a heminested polymerase chain reaction (PCR) assay. A primary focus of infection (respiratory, mucocutaneous, or gynecologic) was present in 22 patients, whereas 5 had no identifiable primary focus of infection. Eighteen patients had systemic infection. Immunohistochemical analysis and PCR can be used for diagnosis of GAS infections in formalin-fixed, paraffin-embedded samples.