ABSTRACT.Large numbers ofPneumocystis carinii(2 × 1010nuclei) were isolated and separated from the lungs of immunosuppressed rats by an enzymatic (collagenase, hyaluronidase and DNase) digestion procedure. The nucleic acid isolated from thisP.carinnii‐enriched preparation was characterized by melting point analysis and RNA‐sizing gels. The GC content ofP. cariniiDNA was approximately 33% while the rat DNA was 41.4%. In addition, RNA isolated from theP. carinii‐enrichedpreparation showed unique ribosomal RNA bands of 3.4 kb and 1.8 kb as compared with uninfected rat lung ribosomal RNA which banded at 4.8 and 1.9 kb. Following isolation and fragmentation by sonication, theP. cariniiDNA fragments were inserted into the vector, λ gt‐11. The resultant library contained 1.1 × 105phage, of which 40–45% hybridized toP. cariniiDNA but no