The Uganda Palo Alto strain of Plasmodium falciparum (FUP) is routinely used as a reference isolate in a number of laboratories. It is one of the few P. falciparum strains that can both be propagated in vivo in monkeys and maintained in culture. The Palo Alto parasites have been characterized for several biochemical and molecular markers, but many of the data reported so far are contradictory. We have analyzed and compared by Southern blotting, PCR and DNA sequencing, several DNA preparations obtained from different FUP lines and from the FCR3 strain. We show here that FUP lines propagated in Saimiri monkeys (FUP/S) and those maintained in culture (FUP/C) for many years in our laboratory differ in the various genetic markers investigated (P190, MSA2, S-Ag, KAHRP, 96 tR, pPFPA rep 20 and pPF 11.1). Therefore, at the present, two genetically unrelated strains of P. falciparum widely distributed over numerous laboratories are designated FUP/Palo Alto. When the Saimiri-propagated FUP/S line was used to initiate an in vitro culture in human red blood cells, no evidence of instability or genetic drift was obtained. The growth rate and genomic characteristics remained constant for several months. Likewise, the FUP/C line was found unchanged after three transfers in Saimiri monkeys. FUP/CP parasites were shown to be genetically closely related to FCR3. In addition, a subline of FUP/C strain selected by repeated flotation on gelatin was found to differ in several characters such as KHARP, P190 and S-antigen genes, which are known to be located on different chromosomes.