Kinetoplast DNA (kDNA) of trypanosomatid protozoa consists of an unusual arrangement of two types of circular molecules catenated into a single network: (1) a few maxicircles that encode various mitochondrial enzyme subunits and rRNA in a cryptic pattern and (2) thousands of minicircles that encode guide RNAs (gRNAs). kDNA is associated with proteins, known as kinetoplast-associated proteins (KAPs), which condense the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasite that displays different kDNA condensation patterns during its complex morphogenetic development. We cloned the T. cruzi gene encoding TcKAP3 (kinetoplast-associated protein 3). TcKAP3 is a single-copy gene that is transcribed into a 1.8-kb mRNA molecule and expressed in all stages of the parasite. Mouse antiserum raised against recombinant TcKPA3 recognized a 21.8kDa protein, which was found, by indirect immunofluorescence and immunoelectron microscopy, to be associated with the T. cruzi kinetoplast. Several features of TcKAP3, such as its small size, basic nature and similarity with KAP3 from the insect trypanosomatid Crithidia fasciculata, are consistent with a role in DNA charge neutralization and condensation. This suggests that this protein is involved in organizing the kDNA network. Gene deletion was used to investigate TcKAP3 function. Here we investigated the T. cruzi KAP3 null mutant, analyzing its fitness during proliferation, differentiation and infectivity.