The ant species Leptothorax tuberum was shown to be predominantly monogynous. Queens usually mate once only but some nests may have a multiply-mated queen or are partially or serially polygynous. As expected from these results, within nest relatedness between workers and between workers and alate queens was found to be high. Almost fifty percent of nests had no nest queen which may indicate high queen mortality, queens leaving to found new nests or nest fragmentation. Observed female investment frequencies (IFo) were not significantly different from those expected on the basis of worker control of sexual production and the relatedness estimates of workers to alate queens and workers to males calculated from isozyme data (IFE). These values were not consistent with queen control. There was no evidence for lower IF(o)s in queenless nests nor for higher IF(o)s in larger nests classified by worker number. When nests were classified by sexual productivity, however, there was a strong rank correlation between productivity and female bias. This is the first study of an ant species to test observed IF against expected IF calculated without inferring between caste relatedness from worker data or pooling of data from different castes.