Alexei Popyrin and Chris O'Connell have been knocked out of the Madrid Open, with Australia's hopes in the men's draw now resting on Alex de Minaur.
But Daria Kasatkina won her first match in Europe since switching allegiance to Australia, beating American Alycia Parks in the women's tournament.
Popyrin's hopes of a deep run crumbled in the round of 64 as the 25th seed posted a 6-4, 7-6 (7/4) loss to Kazakh Alexander Bublik.
O'Connell, who won his opening match earlier in the tournament, fell to third seed Taylor Fritz, with the US Open finalist outplaying the Sydneysider 6-1, 6-4.
The results leave world number seven de Minaur as the sole Australian in the men's draw.
After a first-round bye, de Minaur will face Italian world number 43 Lorenzo Sonego on the main Manolo Santana Stadium court.
Russia-born Kasatkina, the world number 14, was not at her sharpest in the second round but was still far too good for Parks, winning 6-2, 7-5.
Kasatkina, the only Australian representative left in the women's event, is at her strongest on clay and will face a much sterner test against Russian 22nd seed Ekaterina Alexandrova in the round of 32.
Alexandrova defeated Serbian Olga Danilovic 6-3, 6-4 to remain in the tournament.
World number one Aryna Sabalenka, winner in Madrid in 2021 and 2023, overpowered Russia's Anna Blinkova 6-3, 6-4.
Third seed Jessica Pegula beat German Eva Lys 6-2, 6-2 for her tour-leading 27th victory of the year.
But there was a shock with Russian world number 39 Anastasia Potapova upsetting China's Olympic champion, eighth seed Qinwen Zheng, 6-4, 6-4.
AAP
