We describe in situ hybridization protocols using peptide nucleic acid (PNA) as a probe for detecting HIV-1 DNA in virus-infected cells and the subsequent detection of cellular and/or viral proteins. Because a PNA probe of approx 20 bases was sufficiently long to detect a specific target sequence, a conserved sequence of such a short length was easily identified. Therefore, this probe is valuable even to identify quasi-species of HIV-1. In addition, we adopted a catalyzed signal amplification method to amplify weak viral DNA signals; thus, stringent washing was crucial for eliminating false-positive signals. Our double-staining method using PNA-in situ hybridization and subsequent immunostaining enabled the active and inactive proviruses to be distinguished.