Diploid populations of Lumbriculus collected from lakes and sloughs in the Minnesota and Iowa sexually reproduce during the summer months. These worms have reproductive structures including atria, testes, ovaries, sperm sacs, male funnels, female funnels, spermathecae with ventrolateral pores, and protrusible penis sheaths. Histological analysis showed sperm in spermathecae suggesting that cross-fertilization was occurring. The objective of the project was to look for direct evidence of outcrossing. Worms labeled with the fluorescent dye, Hoechst 33342, appeared to transfer sperm to unlabeled worms. We consistently observed fluorescent sperm in homogenates made from reproductive segments of “recipient” worms incubated with labeled worms. However, we were able to detect labeled sperm in the spermathecae of only one of the recipients. We then explored other biological stains to monitor potential transfer of label from “donors” to the spermathecae of “recipient” worms. We utilized a fluorescent dye, rhodamine B, which binds to proteins including those in sperm and seminal fluid and neutral red, a stain taken up by living cells, labeling molecules associated with lysosomes. When worms labeled with either rhodamine B or neutral red were partnered with unlabeled worms, selective transfer of label to the spermathecae of unlabeled worms was observed. These results suggest that out-crossing is occurring in Lumbriculus. Experiments showing that cocoon production by Lumbriculus was dependent on worms being held together rather than individually further support cross fertilization in this freshwater annelid.