The initial appearance of the planktonic foraminiferal genus Hantkenina has been used for about fifty years to recognize the base of the Lutetian and middle Eocene. However, probably as a result of incomplete stratigraphic records, discrepant ranges of Hantkenina have been reported by various investigators at many Eocene sections. Here we report the first complete evolutionary transition from Clavigerinella to Hantkenina, from the northwestern Tethyan deep-water section at Holzhäusl (Salzburg, Austria). A newly discovered species, Hantkenina nov. sp., is the link between Clavigerinella caucasica and Hantkenina mexicana. This finding unequivocally heralds the initial entry of Hantkenina, which is correlated to the upper part of calcareous nannoplankton Subzone NP15b (Sullivania gigas Subzone), to be defined. This indicates a mismatch of ∼4.5 m.y. between the base of the Lutetian at the type locality, which has been placed within Subzone NP14b, and the first appearance datum of Hantkenina. Consequently, the first occurrence of Hantkenina can no longer be used as a marker for the base of the middle Eocene.