Lyme disease is caused by the tick-borne spirochete, Borrelia burgdorferi. It has been documented that B. burgdorferi form aggregates within ticks and during in vitro culture. However, Borrelia aggregates remain poorly characterized, and their functional significance is unknown. Here we have characterized Borrelia aggregates using microscopy and flow cytometry. Borrelia aggregation was temperature, pH, and growth phase dependent. Environmental conditions (high temperature, low pH, and high cell density) favorable for aggregation were similar to the conditions that increased the expression of B. burgdoferi genes, such as outer surface protein C (ospC), that are regulated by the RpoN/RpoS sigma factors. Experiments were conducted to determine if there is a relationship between aggregation and gene regulation through the RpoN/RpoS pathway. ospC Transcript levels were similar between aggregates and free cells. Moreover, no differences were observed in aggregate formation when null mutants of rpoS, rpoN, or ospC were compared to wild-type spirochetes. These results indicated that, despite the similar external signals that promoted aggregation and the RpoN/RpoS pathway, the two processes were not linked at the molecular level. The methods developed here to study B. burgdorferi aggregates will be useful for further studies on spirochete aggregates.