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Twenty (1.4%) of 1,421 adult Ixodes pacificus ticks and 2 (20%) of 10 adult Ixodes neotomae ticks collected in five counties of northern California were found to contain spirochetes by direct immunofluorescence examination of their tissues with a polyvalent conjugate. Borreliae isolated from the tissues of nine of these ticks (I. pacificus, 8; I. neotomae, 1) were identified as Borrelia burgdorferi with specific monoclonal antibodies and characterized further by polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. The isolate from I. neotomae was the first to be characterized from a tick other than I. pacificus in western North America. All strains were relatively homogeneous with respect to the kind of OspA proteins they produced, whereas they were heterogeneous with regard to their OspB proteins and to several low-molecular-weight proteins in the 21,500-to-24,000 region. Significant phenotypic variation was observed among isolates obtained within and between populations of I. pacificus. This investigation nearly doubles the number of isolates of B. burgdorferi that have been characterized from ixodid ticks in the far western United States.