In order to assess the appropriateness of the use of internal transcribed spacer (ITS) sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphisms (T-RFLP). When using the standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF) per strain or cloned sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures. Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR). Modelling results predicted the existence of hairpin loops that would still be present at the extension temperature; it is likely that these loops produced incomplete and single stranded PCR products. We modified the standard T-RFLP procedure by adding the labelled ITS primer in the last two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important tool to study the factors influencing the genetic structure and distribution of these organisms.Key words: cyanobacteria, DGGE, T-RFLP, ITS, secondary structure. RESUMENCon el fin de evaluar la utilización de secuencias del espaciador interno transcrito (ITS) en estudios de genética de población de cianobacterias marinas, se amplificó y clonó la secuencia del gen ARNr 16S junto a la región espaciadora 16S-23S ARNr de seis cepas de Prochlorococcus y Synechococcus. Se analizaron los amplicones del ITS por electroforesis en gel de gradiente de desnaturalización (DGGE) y por polimorfismos de longitud de fragmentos de restricción terminal (T-RFLP). Al aplicar los métodos estándares de estas técnicas, se obtuvo más de una banda o fragmento de restricción terminal (T-RF) por cepa o secuencia clonada. Informes en la literatura han sugerido que estas anomalías podrían ser atribuidas a la formación de estructuras secundarias. Por consiguiente, la estructura secundaria de las secuencias de ITS de las cepas de Prochlorococcus y Synechococcus fue modelada a las diferentes temperaturas que se utilizaron durante la reacción en cadena de polimera...