The foliage of Pinus strobus (eastern white pine), as that of all other conifers examined, is occupied by endophytic fungi, the most frequent of which is Lophodermium nitens. The number and extent of endophytic infections and the genetic relationship of individual isolates within living needles as well as their relationship to isolates from forest floor needles is unknown. To examine these and related questions, forest floor isolates and foliar endophytes from needle segments were obtained for ribosomal DNA sequencing and randomly amplified polymorphic DNA (RAPD) analysis. Molecular and morphological data were compared and infection frequency determined as a function of position along the needle. Ribosomal DNA sequences of foliar and ascospore isolates showed high levels of genetic similarity (>97% identity) for the internal transcribed spacer region. RAPD profiles were able to distinguish ascospore siblings from non-siblings, and also revealed that many needle isolates belonged to the same genotype as adjacent neighbour isolates, as would be expected from mycelial spread within the needle. Morphotype evaluation and RAPD profiles showed similar patterns: identical morphotypes grouped together and showed little or no genetic difference under RAPD analysis. Both morphological and molecular data indicated that the majority of infections were contained within 1 mm needle segments but could extend to about 4 mm in length. Infection frequency increased along the length of the needle from the proximal (shoot) end to the distal tip, with markedly higher rates in the distal quarter. Thus, endophytic infections of L. nitens in white pine needles consist of many localized, discrete infections, originating from ascospores and differentially distributed along the length of the needle. In the course of this work, it was found that GenBank accession no. AF203470 under the name Meloderma desmaszieresii appeared not to be that species but L. nitens on the basis of the ITS sequences.